WO1998042830A2 - Nouvelles compositions de genes noey2 et leurs utilisations - Google Patents

Nouvelles compositions de genes noey2 et leurs utilisations Download PDF

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Publication number
WO1998042830A2
WO1998042830A2 PCT/US1998/005723 US9805723W WO9842830A2 WO 1998042830 A2 WO1998042830 A2 WO 1998042830A2 US 9805723 W US9805723 W US 9805723W WO 9842830 A2 WO9842830 A2 WO 9842830A2
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Prior art keywords
nucleic acid
cell
noey2
dna
polypeptide
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PCT/US1998/005723
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English (en)
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WO1998042830A3 (fr
Inventor
Yinhua Yu
Fengji Xu
Robert C. Bast, Jr.
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Board Of Regents, The University Of Texas System
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Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Priority to EP98911977A priority Critical patent/EP0988376A2/fr
Priority to US10/166,325 priority patent/US7183401B1/en
Priority to JP54590098A priority patent/JP2001517954A/ja
Priority to CA002284685A priority patent/CA2284685A1/fr
Priority to AU65805/98A priority patent/AU6580598A/en
Publication of WO1998042830A2 publication Critical patent/WO1998042830A2/fr
Publication of WO1998042830A3 publication Critical patent/WO1998042830A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates generally to the fields of oncology and molecular biology. More particularly, it concerns nucleic acid segments isolated from human chromosome lp31, which encode a novel tumor suppressor protein, designated NOEY2.
  • NOEY2 a novel tumor suppressor protein
  • Various methods for making and using NOEY2 DNA segments, DNA segments encoding synthetically-modified NOEY2 proteins, and native and synthetic tumor suppressor proteins are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods for identifying NOEY2-related tumor suppressor polynucleotides and polypeptides, and methods for treating tumors, and in particular, ovarian and breast-related cancers.
  • Oncogenesis was described by Foulds (1958) as a multistep biological process, which is presently known to occur by the accumulation of genetic damage.
  • the multistep process of tumorigenesis involves the disruption of both positive and negative regulatory effectors (Weinberg, 1989). Defects leading to the development of retinoblastoma have been linked to a tumor suppressor gene (Lee et al. , 1987), and a variety of oncogenes and other tumor suppressors have been identified in a host of malignancies.
  • Unfortunately there remains an inadequate number of treatable cancers, and the effects of cancer are catastrophic — over half a million deaths per year in the United States alone.
  • Tumor suppressor proteins function to negatively regulate cell cycle processes, preventing the uncontrolled growth exhibited by cancerous cells. Tumor suppressor proteins function by transcriptional regulation of key genes involved in cellular growth and division. Unlike protooncogenes, for which activation is required for the initiation of cancerous growth, inactivation of tumor suppressors leads to cancer. In normal diploid cells, two copies of the gene encoding a particular tumor suppressor protein are present. The "two hit" hypothesis states that mutation or inactivation of both individual copies of the gene is required for the onset of cancerous growth. Mutations may include deletions, alterations to transcription levels, single or multiple coding changes, and truncations.
  • Inherited mutations in a single tumor suppressor gene typically leads to a recessive heterozygous phenotype. Cells remain phenotypically normal until mutation of the second, wild type copy of the gene. Familial inheritance of a mutant copy of a tumor suppressor gene does render the individual more prone to cancer, as inactivation of the single normal gene is sufficient to initiate cancer.
  • the two most heavily studied tumor suppressor genes are the retinoblastoma gene Rb and the p53 gene.
  • the retinoblastoma gene Rb was the first discovered tumor suppressor gene.
  • the Rb protein is a 110 kDa nuclear phosphoprotein that reduces the growth rate of cells, and mutations in this protein have been found in breast, prostate, and small cell lung carcinomas.
  • the Rb protein binds to DNA, but without any sequence specificity.
  • Rb effects its regulatory function through the formation of complexes with transcription factors.
  • Rb has been shown to interact with transcription factor E2F, which binds to the promoters of cellular genes such as DNA polymerase ⁇ and ribonucleotide reductase. Heterozygous individuals inherit one defective and one normal copy of the Rb gene. Upon inactivation of the second, functional copy of the gene, cancerous growth initiates.
  • a wide range of mutations including deletions, duplications, and point mutations have been found to lead to inactivation of the Rb protein.
  • the p53 tumor suppressor has been found to be mutated in about 60% of human cancerous growths, making p53 the most commonly mutated gene in human cancers.
  • Wild type p53 protein binds to DNA and is believed to function as a transcriptional regulator.
  • the p53 protein also binds to the mdm2 protein, commonly expressed at high levels in tumors. Levels of wild type p53 protein increases upon subjecting a cell to radiation or chemical agents which damage DNA.
  • the p53 protein has been implicated in DNA repair mechanisms that prevent the duplication of damaged or altered DNA. It has been further speculated that p53 protein prevents the cell from entering S phase until the complete repair of damaged DNA has been achieved.
  • the p53 protein is normally found at low, often undetectable levels in cells, and has a short cellular half life. Mutant p53 proteins often are more resistant to degradation, and are present at immunohistochemically detectable levels in cancerous cells. Most p53 gene mutations occur in four highly conserved regions. Mutant p53 proteins are dominant inactivators, by their ability to bind to and inactivate wild type p53 protein.
  • the ovary is the fifth most common site of cancer among American women and ovarian neoplasms constitute the fourth leading cause of cancer death.
  • Ovarian cancer affects 22,000 women in the United States each year and causes some 14,000 deaths annually.
  • Approximately 90% of ovarian cancers arise from epithelial cells that cover the ovarian surface or that line inclusion cysts.
  • Ovarian cancers exhibit a distinctive pattern of metastasis.
  • ovarian cancer can metastasize to pelvic and retroperitoneal lymph nodes and can spread hematogenously to distant sites. More frequently, however, ovarian cancer cells spread over the surface of the peritoneal cavity, forming multiple nodules on the parietal and visceral peritoneum.
  • Blockade of diaphragmatic lymphatics and increased transudation of fluid produce accumulation of ascites fluid that contains varying numbers of tumor cells.
  • Abdominal distention by malignant ascites is a frequent mode of clinical presentation.
  • Early stage ovarian cancer rarely produces symptoms and at present there is no proven strategy for early detection.
  • tumor cells have metastasized beyond the ovaries at presentation.
  • Initial clinical management generally involves cytoreductive surgery and drainage of ascites, providing large amounts of tissue for study. Following removal of as much tumor as possible, cytotoxic chemotherapy is generally administered.
  • Introduction of platinum based compounds and the taxane derivatives has improved median survival of patients with advanced disease, but the five year survival rate is still only 28% for all stages and has not improved in the last several decades.
  • Breast cancer is the most common form of cancer among women, affecting about one in eight women. Approximately 185,700 new cases are diagnosed in the U. S. annually, and breast cancer is responsible for about 44,560 deaths in the U. S. per year. While predominantly observed in women, 1 ,400 cases of breast cancer are diagnosed annually in men, and 260 men die of breast cancer per year. Breast cancer first manifests itself as a painless lump, detectable by self-examination and clinical breast exams including mammograms. Commonly, growth initiates in the lining of the ducts or in the lobules of the breast. Current clinical treatments include mastectomy (removal of the entire breast) or lumpectomy (removal of the tumor and surrounding tissue) for localized tumors.
  • Chemotherapy, radiotherapy, or hormone-blocking therapy may be further used to control cancerous cells.
  • Breast cancer cells can metastasize to the lymph nodes, skin, lungs, liver, brain, or bones. Metastasis may occur early or late in the disease progression, although typically metastasis occurs once the cancerous growth reaches a size of about 20 mm. Metastasis is achieved by cells breaking away from the parental mass and entering either the bloodstream or the lymphatic system.
  • BRCAl mutations are present at between 1 in 300 to 1 in 800 females. In the BRCAl gene, over 200 different mutations have been discovered to date. The mutations observed are not localized to a single region, further complicating genetic analysis. Greater than 80% of the observed mutations result in a truncated form of the BRCAl protein. Individuals with familial hereditary BRCAl possess one normal and one mutant form of the gene, and are therefore much more likely to develop breast cancer.
  • BRCA2 has been identified on chromosome 13q through linkage analysis of 15 breast cancer families that did not demonstrate BRCAl linked breast cancer. Unlike BRCAl mutations, BRCA2 does not substantially elevate the risk of ovarian cancers.
  • the BRCA2 gene encodes a protein of 3,418 amino acids, many of which are acidic or basic. Most mutations observed involve base deletions that alter the reading frame, and result in a premature truncation of the protein. BRCAl and BRCA2 account for about 45% of familial inherited breast cancers each, leaving 10% for one or more additional genes. Interestingly, all male breast cancers appear to be due to mutations in the BRCA2 gene.
  • tumor suppressor genes There are relatively few tumor suppressor genes whose mutations have been shown to correlate with the presence of cancerous cells. Those that have been characterized typically have many possible types and positions of mutations, complicating genetic analyses and the prediction of cancer predisposition. Therefore, what is lacking in the prior art is the identification and characterization of novel tumor suppressor genes, and identification of the role of the proteins encoded by such genes in cancer diagnosis and treatment. Such genes, in combination with improved genetic testing, and improved correlation of specific genetic mutations with particular cancer susceptibility are needed to facilitate early and effective treatment of these proliferative diseases.
  • the present invention overcomes these and other limitations in the prior art by providing a novel tumor suppressor protein (designated NOEY2) (SEQ ID NO:2) and the gene which encodes it (designated NOEY2).
  • NOEY2 tumor suppressor protein
  • This gene is expressed in normal ovarian surface epithelial cells, but consistently absent or down-regulated in ovarian cancer cells.
  • the invention provides unique polynucleotide sequences which comprise the cDNA (SEQ ID NO:l) and the genomic DNA (SEQ ID NO:5) encoding this NOEY2 tumor suppressor.
  • a further objective of the invention is to provide polynucleotide segments comprising all or parts of a gene encoding NOEY2.
  • Polynucleotide probes and primers specific for these NOEY2 genes also represent important compositions provided by the invention. It is a further objective of the invention to provide antibodies specific for NOEY2, methods for identifying NOEY polypeptide and polynucleotide compositions, methods for producing such compositions, and methods for using these compositions in a variety of diagnostic and therapeutic regimens. The invention also provides methods and compositions for the detection of NOEY2 compositions in biological and clinical samples, and methods for regulating the proliferation of tumor cells in vitro and in vivo.
  • the invention provides an isolated and purified amino acid segment comprising a NOEY2 tumor suppressor protein (SEQ ID NO:2) comprising the amino acid sequence of SEQ ID NO:2.
  • SEQ ID NO:2 tumor suppressor protein (SEQ ID NO:2) comprising the amino acid sequence of SEQ ID NO:2.
  • the coding region for the NOEY2 polypeptide is from nucleotide 150 to 833 of SEQ ID NO:l, (the cDNA for NOEY2).
  • the genomic DNA sequence is presented in SEQ ID NO:5.
  • the NOEY2 protein exhibits tumor suppressor activity which is related to cyclin DI promoter inhibition.
  • methods for making and using this protein, derivatives and mutants thereof, and antibodies directed against these proteins are also disclosed.
  • the invention provides an isolated and purified nucleic acid segment comprising the NOEY2 gene which encodes the NOEY2 tumor suppressor protein disclosed herein.
  • the nucleotide sequence of an exemplary NOEY2 gene is given from position 150 to position 833 of SEQ ID NO: l (NOEY2 cDNA) and SEQ ID NO:5 (NOEY2 genomic).
  • methods for making, using, altering, mutagenizing, assaying, and quantitating these nucleic acid segments are also disclosed. Also disclosed are diagnostic methods and assay kits for the identification and detection of related NOEY2 gene sequences in a variety of in vitro and in vivo methodologies.
  • an animal cell such as a human or other animal cell, that comprises a NOEY2 polypeptide or polynucleotide.
  • the cell is an ovarian surface epithelial cell that produces a tumor suppressor polypeptide of approximately 26-kDa, and that is identical to, or substantially homologous with, the NOEY2 polypeptide identified in SEQ ID NO:2.
  • a further aspect of the present invention is a vector (such as a plasmid, cosmid, virus, phagemid, or the like), that includes within its nucleotide sequence a nucleic acid segment that comprises one or more NOEY2 genes, or portions thereof.
  • a vector is comprised within a transformed host cell.
  • the transformed host cell may be a bacterial, animal, fungal, or plant cell, and may be comprised within a transgenic animal, or may be comprised within a culture of bacteria, yeast, fungus, animal or plant cells.
  • a monoclonal antibody that binds immunologically to a tumor suppressor designated as NOEY2.
  • the antibody may be non-cross reactive with other human polypeptides, or it may bind to non-human NOEY2, but not to human NOEY2.
  • the antibody may further comprise a detectable label, such as a fluorescent label, a chemiluminescent label, a radiolabel or an enzyme. Also encompassed are hybridoma cells and cell lines producing such antibodies.
  • a polyclonal antisera antibodies of which bind immunologically to a tumor suppressor designated as NOEY2.
  • the antisera may be derived from any animal, but preferably is from an animal other than a human.
  • Preferred antigens for the preparation of such sera include a NOEY2 polypeptide isolated from a human, rat, goat, rabbit, pig, horse, cat, dog, hamster, monkey or other such animal cell line.
  • Preferred hosts for the preparation of a polyclonal antisera specific for NOEY2 include animals such as rabbits, goats, and other such animals.
  • compositions which comprise one or more of the NOEY2 compositions disclosed herein.
  • Such compositions may include NOEY2 or NOEY2-derived polypeptides, polynucleotides, antibodies, antisera, antigens, peptide epitopes, protein fusions, peptides and the like.
  • a method of diagnosing a cancer comprising the steps of (a) obtaining a sample from a subject; and (b) determining the expression a functional NOEY2 tumor suppressor in cells of the sample.
  • the cancer is ovarian or breast cancer, although the cancer may also be brain, lung, liver, splenic, renal, lymphatic, intestinal, pancreatic, leukemia, colon, stomach, endometrial, prostate, testicular, skin, head and neck, esophageal, bone marrow or blood cancer.
  • the cancer is ovarian cancer or breast cancer.
  • the sample is a cell, cell culture, tissue or fluid sample, and may be of clinical or non-clinical origin.
  • the method involves assaying for a nucleic acid from the sample.
  • the method may further comprise subjecting the sample to conditions suitable to amplify the nucleic acid.
  • the method may comprise contacting the sample with an antibody that binds immunologically to a NOEY2, for example, in an ELISA.
  • the comparison regardless of format, may include comparing the expression of NOEY2 with the expression of NOEY2 in non-cancer samples, for example in normal ovarian epithelial cells.
  • the comparison may look at levels of NOEY2 expression.
  • the comparison may involve evaluating the structure of the NOEY2 gene, protein or transcript.
  • Such formats may include sequencing, wild-type oligonucleotide hybridization, mutant oligonucleotide hybridization, SSCP, PCRTM and/or RNase protection.
  • Particular embodiments include evaluating wild-type or mutant oligonucleotide hybridization where the oligonucleotide is configured in an array, on a chip, on a wafer, or in a microtiter dish.
  • a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a tumor suppressor designated NOEY2 under conditions permitting the uptake of the tumor suppressor by the tumor cell.
  • the tumor cell may be derived from an animal organ or tissue such as brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, or bone marrow.
  • the tumor cell is derived from an ovarian or breast cancer cell.
  • the phenotype may be selected from proliferation, migration, contact inhibition, soft agar growth or cell cycling.
  • the tumor suppressor polypeptide may be provided in a pharmaceutical formulation, encapsulated in a liposome, nanocapsule or other lipid particle, or may be carrier-free.
  • a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a nucleic acid (a) encoding a NOEY2 tumor suppressor and (b) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the tumor suppressor, under conditions permitting the uptake of the nucleic acid by the tumor cell.
  • the phenotype may be proliferation, migration, contact inhibition, soft agar growth or cell cycling.
  • the tumor suppressor-encoding nucleic acid may be provided in a pharmaceutical formulation, encapsulated in a liposome, nanocapsule or other lipid particle, or may be carrier-free.
  • the nucleic acid is comprised within a viral vector such as retrovirus, adenovirus, adeno-associated virus, vaccinia virus and herpesvirus, it may also be encapsulated in a viral particle.
  • the invention further provides a method of inhibiting cellular proliferation, comprising providing to a cell a composition comprising a NOEY2 tumor suppressor polypeptide, or a NOEY2 polynucleotide which expresses a NOEY2 polypeptide in a pharmaceutically acceptable vehicle.
  • the present invention also provides a method of inhibiting tumor proliferation, comprising providing to a tumor cell a composition comprising a NOEY2 tumor suppressor polypeptide or a NOEY2 gene which expresses the NOEY2 protein in a pharmaceutically acceptable vehicle.
  • a method for treating cancer comprising the step of contacting a tumor cell within a subject with a tumor suppressor designated NOEY2 under conditions permitting the uptake of the tumor suppressor by the tumor cell.
  • the method may involve a human subject.
  • a method for treating cancer comprising the step of contacting a tumor cell within a subject with a nucleic acid (a) encoding a NOEY2 tumor suppressor and (b) a promoter active in the tumor cell, wherein the promoter is operatively linked to the region encoding the tumor suppressor, under conditions permitting the uptake of the nucleic acid by the tumor cell.
  • the subject is preferably an animal, and most preferably a human.
  • transgenic mammal in which both copies of the native NOEY2 gene are interrupted or replaced with another gene.
  • a method of determining the stage of cancer comprising the steps of (a) obtaining a sample from a subject; and (b) determining the expression a functional NOEY2 polypeptide in cells of the sample.
  • the cancer is preferably a breast cancer or ovarian cancer.
  • the determining may comprise assaying for a NOEY2 nucleic acid or polypeptide in the sample.
  • a method of predicting tumor metastasis comprising the steps of (a) obtaining a sample from a subject; and (b) determining the expression a functional NOEY2 polypeptide in cells of the sample.
  • the cancer may be distinguished as metastatic and non-metastatic.
  • the determining may comprise assaying for a NOEY2 nucleic acid or NOEY2 polypeptide in the sample.
  • a method of screening a candidate substance for anti-tumor activity comprising the steps of (a) providing a cell lacking functional NOEY2 polypeptide; (b) contacting the cell with the candidate substance; and (c) determining the effect of the candidate substance on the cell.
  • the cell may be a tumor cell, for example, a tumor cell having a mutation in the coding region of NOEY2.
  • the mutation may be a deletion mutant, an insertion mutant, a frameshift mutant, a nonsense mutant, a missense mutant or splice mutant.
  • the determining may comprise comparing one or more characteristics of the cell in the presence of the candidate substance with characteristics of a cell in the absence of the candidate substance.
  • the characteristic may be NOEY2 expression, phosphatase activity, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression and tissue invasion.
  • the candidate substance may be a chemotherapeutic or radiotherapeutic agent or be selected from a small molecule library.
  • the cell may be contacted in vitro or in vivo.
  • the present invention concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the whole or a portion of the novel peptide disclosed herein.
  • the NOEY2 gene (position 150 to position 833 of SEQ ID NO:l and SEQ ID NO: 5) encodes a NOEY2 polypeptide having the contiguous amino acid sequence shown in SEQ ID NO:2.
  • DNA segments from the present invention will find particular utility. For example, those segments that encode all or portions of the NOEY2 polypeptide, or subunits, functional domains, and the like of NOEY2 and NOEY2 -related polypeptides, or those segments that comprise one or more NOEY2 promoter or enhancer regions will be useful in a variety of diagnostic, and therapeutic regimens.
  • Such DNA segments may be native DNA segments isolated using molecular biological methods, or alternatively, such segments may be mutagenized segments, or even segments which have been synthesized in vitro either partially or entirely, using chemical synthesis methods that are well-known to those of skill in the art.
  • the term "DNA segment” refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
  • a DNA segment encoding a tumor suppressor protein or peptide refers to a DNA segment that contains a NOEY2 polypeptide-coding sequence yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term "DNA segment", are DNA segments comprising entire NOEY2 genes and/or promoter regions, as well as all partial and smaller fragments and subfragments isolatable from such entire gene-comprising segments, and also recombinant vectors (such as plasmids, cosmids, phagemids, phage, viruses, and the like) which comprise one or more of the NOEY2-specific polynucleotide sequences of the invention.
  • the segments may comprise gene sequences which are identical to, or substantially homologous with, a contiguous nucleotide sequence from about position position 150 to about position 833 of SEQ ID NO:l or SEQ ID NO: 5, or gene sequences which encode polypeptides which are identical to, or substantially biologically-functionally equivalent to, the polypeptide disclosed in SEQ ID NO:2.
  • a DNA segment comprising an isolated or purified tumor suppressor protein- encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit.
  • this functional term includes not only genomic sequences, including extrachromosomal DNA sequences, but also operon sequences and/or engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case, a NOEY2 tumor suppressor gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.
  • the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a NOEY2 polypeptide that includes within its amino acid sequence an at least ten amino acid contiguous sequence from SEQ ID NO:2, and more preferably still, a polypeptide that includes within its amino acid sequence a sequence essentially as set forth in SEQ ID NO:2.
  • a DNA segment comprises a gene encoding the amino acid sequence of SEQ ID NO:2, and more preferably still, comprises a polynucleotide which is identical to, or substantially homologous with, the DNA sequence of SEQ ID NO:l or SEQ ID NO:5.
  • sequences essentially as set forth in SEQ ID NO:2 means that the sequence substantially corresponds to a portion of the sequence of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of SEQ ID NO:2 will be sequences that are "essentially as set forth in SEQ ID NO:2.”
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments may be prepared that include a short contiguous stretch encoding the whole or a portion of the peptide sequence disclosed in SEQ ID NO:2, or that are identical to or complementary to DNA sequences which encode the peptide disclosed in SEQ ID NO:2, and particularly the DNA segment disclosed in either of SEQ ID NO:l or SEQ ID NO:5.
  • DNA sequences such as about 14 nucleotides, and that are up to about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
  • intermediate lengths means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc. ; 21, 22, 23, etc. ; 30, 31, 32, etc. ; 50, 51, 52, 53, etc. ; 100, 101, 102, 103, etc. ; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and including sequences of about 10,000 nucleotides and the like.
  • this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of SEQ ID NO:2, including the DNA sequence which is particularly disclosed in SEQ ID NO:l and SEQ ID NO:5.
  • Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
  • DNA segments of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site- directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
  • fusion proteins and peptides e.g., where the peptide- coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • Recombinant vectors form further aspects of the present invention.
  • Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter.
  • the promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5' non- coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
  • nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA segment of SEQ ID NO:l or SEQ ID NO:5 will find particular utility.
  • nucleic acid probes to specifically hybridize to tumor suppressor protein-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample.
  • sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to the DNA sequence of SEQ ID NO:l or SEQ ID NO:5, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting.
  • hybridization probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion.
  • Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patents 4,683,195 and 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.
  • one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating tumor suppressor protein-encoding DNA segments.
  • hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
  • an appropriate means such as a label
  • a wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.
  • the invention also discloses and claims a composition comprising a NOEY2 tumor suppressor protein.
  • the composition may comprises one or more host cells which express a NOEY2 tumor suppressor protein, recombinant host cells expresses the protein, cell suspensions, extracts, inclusion bodies, or tissue cultures or culture extracts which contain the NOEY2 protein, culture supernatant, disrupted cells, cell extracts, lysates, homogenates, and the like.
  • the compositions may be in aqueous form, or alternatively, in dry, semi -wet, or similar forms such as cell paste, cell pellets, or alternatively freeze dried, powdered, lyophilized, evaporated, or otherwise similarly prepared in dry form.
  • the tumor suppressor proteins may be purified, concentrated, admixed with other reagents, or processed to a desired final form.
  • the composition will comprise from about 1 % to about 90% by weight of the tumor suppressor protein, and more preferably from about 5% to about 50% by weight.
  • the tumor suppressor protein compositions of the invention may be prepared by a process which comprises the steps of culturing a host cell which expresses a NOEY2 tumor suppressor protein under conditions effective to produce such a protein, and then obtaining the protein from the cell.
  • the obtaining of such a tumor suppressor protein may further include purifying, concentrating, processing, or admixing the protein with one or more reagents.
  • the NOEY2 tumor suppressor protein is obtained in an amount of from between about 1% to about 90% by weight, and more preferably from about 5% to about 70% by weight, and even more preferably from about 10% to about 20% to about 30%, or even to about 40% or 50% by weight.
  • the invention also relates to a method of preparing a tumor suppressor protein composition.
  • a method generally involves the steps of culturing a host cell which expresses a NOEY2 tumor suppressor protein under conditions effective to produce the protein, and then obtaining the protein so produced.
  • the cell is an NIH3T3 cell, or any recombinant host cell which contains a NOEY2-encoding DNA segment.
  • the recombinant plasmid vectors of the invention may be used to transform other suitable bacterial or eukaryotic cells to produce the tumor suppressor protein of the invention.
  • Eukaryotic host cells including NIH3T3, COS7, and CAOV3, as well as yeast cells are contemplated to be particularly useful in the preparation of the NOEY2 protein.
  • prokaryotic host cells including Gram-negative cells such as E. coli, Pseudomonas spp. and related Enterobacteraceae and the like are all contemplated to be useful in the preparation of the tumor suppressor proteins of the invention. In such embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a tumor suppressor protein or peptide in its natural environment.
  • Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, or eukaryotic cell.
  • Preferred eukaryotic cells are animal cells, with mammalian cells, particularly human cells, being most preferred.
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989.
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
  • Pichia expression vector system Pharmacia LKB Biotechnology
  • DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful.
  • Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from SEQ ID NO:2.
  • the present invention provides methods for producing a transgenic cell, and in particular a plant or animal cell which expresses a nucleic acid segment encoding the novel NOEY2 tumor suppressor protein of the present invention.
  • the process of producing transgenic cells is well-known in the art.
  • the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operatively linked to a coding region that encodes a NOEY2 tumor suppressor protein.
  • a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo.
  • the invention also provides for the expression of tumor suppressor protein antisense mRNA.
  • antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.
  • the invention encompasses an animal cell which has been transformed with a nucleic acid segment of the invention, and which expresses a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein.
  • transgenic host cell is intended to refer to a host cell, either prokaryotic or eukaryotic, that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed host cell, such as genes which may normally be present in the non-transformed cell but which one desires to either genetically engineer or to have altered expression.
  • the genome of a transgenic host cell of the present invention will have been augmented through the stable introduction of a NOEY2 transgene, either native NOEY2, or synthetically modified or mutated NOEY2.
  • more than one transgene will be incorporated into the genome of the transformed host cell.
  • more than one tumor suppressor protein-encoding DNA segment is incorporated into the genome of such a cell.
  • the introduction of the transgene into the genome of the host cell results in a stable integration wherein the progeny of such cells also contain a copy of the transgene in their genome.
  • a preferred gene which may be introduced includes, for example, a tumor suppressor protein-encoding a DNA sequence, and particularly one or more of the NOEY2 or NOEY2-like tumor suppressor proteins disclosed herein.
  • Highly preferred nucleic acid sequences are those which have the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:5, or biologically- functional equivalents thereof, sequences which hybridize to the sequence of SEQ ID NO:l or SEQ ID NO:5, or sequences which encode the amino acid sequence of SEQ ID NO:2, or sequences which encode a biologically functional equivalent protein of SEQ ID NO:2, or any of those sequences which have been genetically engineered to alter, modify, change, decrease or increase the suppressor activity or specificity of the tumor suppressor protein in such a transformed host cell.
  • Vectors including plasmids, cosmids, phage, phagemids, BACs (bacterial artificial chromosomes), YACs (yeast artificial chromosomes), and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene- derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed tumor suppressor proteins.
  • These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired.
  • the DNA segment or gene may encode either a native or modified tumor suppressor protein, which will be expressed in the resultant recombinant cells, and/or which will impart a desired phenotype to the transformed host cell.
  • the inventors contemplate the use of antibodies, either monoclonal or polyclonal which specifically bind to one or more of the NOEY2 polypeptides disclosed herein.
  • Means for preparing and characterizing antibodies are well known in the art (See, e.g, Harlow and Lane, 1988; incorporated herein by reference).
  • the methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified tumor suppressor protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 to 2 x 10 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS- 1-Ag4-1
  • P3-NS- 1-Ag4-1 Another preferred murine myeloma cell
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody -producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al, 1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 " to
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single- clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. 2.7 NOEY2 POLYPEPTIDE SCREENING METHODS AND IMMUNODETECTION KITS
  • the present invention also provides compositions, methods and kits for screening samples suspected of containing a NOEY2 polypeptide or a NOEY2 polynucleotide that encodes such a tumor suppressor protein.
  • the invention provides compositions, methods and kits for screening samples suspected of containing tumor suppressor proteins or genes encoding tumor suppressor proteins which are functionally equivalent to, or substantially homologous to, the NOEY2 tumor suppressor protein disclosed herein.
  • Such screening may be performed on samples such as transformed host cells, clinical or laboratory samples suspected of containing or producing such a polypeptide or nucleic acid segment.
  • a kit can contain a novel nucleic acid segment or an antibody of the present invention.
  • the kit can contain reagents for detecting an interaction between a sample and a nucleic acid or an antibody of the present invention.
  • the provided reagent can be radio-, fluorescently- or enzymatically-labeled.
  • the kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.
  • the reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder.
  • the liquid solution is an aqueous solution.
  • the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide.
  • the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
  • the present invention concerns immunodetection methods and associated kits. It is proposed that the tumor suppressor proteins or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect tumor suppressor proteins or tumor suppressor protein-related epitope-containing peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.
  • immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches.
  • the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like.
  • immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
  • a label such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
  • a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
  • any sample suspected of comprising either a tumor suppressor protein or peptide or a tumor suppressor protein-related peptide or antibody sought to be detected may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.
  • the present invention contemplates the preparation of kits that may be employed to detect the presence of tumor suppressor proteins or related peptides and/or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing tumor suppressor proteins or peptides.
  • kits in accordance with the present invention will include a suitable tumor suppressor protein, peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent.
  • the immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand.
  • Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label.
  • a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
  • the container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted.
  • the kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained. 2.8 NOEY2-DERIVED EPITOPIC SEQUENCES
  • the present invention is also directed to NOEY2 protein or peptide compositions, free from total cells and other peptides, which comprise a purified NOEY2 protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-tumor suppressor protein antibodies.
  • the invention concerns epitopic core sequences derived from NOEY2 and NOEY2-derived proteins or peptides.
  • the term "incorporating an epitope(s) that is immunologically cross- reactive with one or more anti-tumor suppressor protein antibodies” is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a tumor suppressor protein or polypeptide.
  • the level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the tumor suppressor protein or polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen.
  • Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
  • NOEY2 immunodominant epitopes and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter.
  • Hopp as taught in U. S. Patent 4,554,101, inco ⁇ orated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity.
  • the methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, e.g., Jameson and Wolf, 1988; Wolf e/ al., 1988; U. S. Patent Number 4,554,101).
  • amino acid sequence of these "epitopic core sequences” may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
  • Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to about 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic tumor suppressor protein-derived peptides will provide advantages in certain circumstances, for example, in the preparation of immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibihty of production, and advantageous biodistribution.
  • An epitopic core sequence is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on the tumor suppressor protein-directed antibodies disclosed herein. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
  • the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • the identification of epitopic core sequences is known to those of skill in the art, for example, as described in U. S.
  • Patent 4,554,101 incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity.
  • numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al., 1988).
  • Computerized peptide sequence analysis programs e.g., DNAStar® software, DNAStar, Inc., Madison, WI may also be useful in designing synthetic peptides in accordance with the present disclosure.
  • Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosy stems Model 430 A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
  • FIG. 1A Complete NOEY2 cDNA nucleotide sequence (SEQ ID NO:l) and the deduced amino acid sequence (SEQ ID NO:2) of the NOEY2 protein. An asterisk indicates the stop codon.
  • FIG. IB Continuation of sequence of Complete NOEY2 cDNA nucleotide sequence (SEQ ID NO:l) and the deduced amino acid sequence (SEQ ID NO:2) of the NOEY2 protein.
  • FIG. 2 The pairwise amino acid sequence comparisons of NOEY2 with Ras and Rap family members. Four GDP/GTP binding domains and the CAAX motif are indicated by underlining. The bold type indicates residues conserved in nearly all GTPases.
  • FIG. 3A Northern blot analysis of NOEY2 in cells and tissues.
  • NOEY2 cDNA probe was labeled with P-dCTP by random primer.
  • Fifteen micrograms of total cellular RNA was separated in 1.2% formaldehyde-agarose gels and immobilized on a Hybond-N + membrane (Amersham) by standard capillary transfer and UV crosslinking, and then prehybridized and hybridized to NOEY2 probe in 50% formamide, IX SSC, 10X Denhardt's solution, 10 mM EDTA, 0.1% SDS and 300 ⁇ g/ml denatured salmon sperm DNA at 42°C for 24 h.
  • OSE cells (lane 1 to 3) and ovarian cancer cell lines (lane 4 to 9).
  • the primary normal OSE cells culture were obtained by gently scraping the surface of the ovaries which were from the patients undergoing surgery for nonmalignant gynecological diseases.
  • the scraped epithelial cells were cultured in OSE medium (MCDB 105/199 medium supplemented with 15% fetal calf serum and 10 ng/ml Epidermal Growth Factor).
  • OSE cells were stained by antibody against cytokeratins.
  • Ovarian cancer cell lines culture was as described before (Kruk et al. , 1990).
  • FIG. 3B Normal breast epithelial cells (NBE) cancer cell lines (lanes 1 to 4) and breast cancer cell lines (lanes 5 to 12).
  • NBE normal breast epithelial cells
  • FIG. 3C OSE cells (lane 1 to 3) and ovarian cancer patients' ascites cells (lane 4 to 9).
  • Ovarian cancer cells primary cultures were obtained by separating tumor cells from ovarian cancer patients' ascites. Patients' ascites cells were thawed, centrifuged and resuspended in 2 ml stock Iso-osmotic Percoll (SIP) and placed in 15 ml tubes, then using Pasteur pipettes, 2 ml each of five different diluted SIP (the densities were 1.070, 1.058, 1.047, 1.035 and 1.023) were carefully layered to form a gradient. Gradients were centrifuged at 1500 rpm for 20 min using a swinging bucket centrifuge.
  • SIP Iso-osmotic Percoll
  • ascites samples contain tumor cells that fraction near the second and third interfaces of the percoll gradient. H & E stains were performed to check the composition of the fractions. After processing ascites by percoll density gradient, further purification was obtained using magnetic beads coated with CD45. The purified cancer cells were stained by five monoclonal antibodies which specifically reacted with ovarian cancer antigens.
  • FIG. 3D Multiple human tissues (Clonetech). 2 ⁇ g poly-RNA each line. Hybridization was done according to the methods of the manufacturer.
  • FIG. 3E Western blot of 26-kDa protein, NIH3T3-NOEY2 (positive control (lane
  • FIG. 4A NOEY2-induced growth inhibition of ovarian and breast cancer cell lines. Colony formation after NOEY2 cDNA transfection. (a. Carrier DNA only; b. pcDNA3 vector only; c. pcDNA3 vector with NOEY2 in the antisense orientation; d. pcDNA3 vector with NOEY2 in the sense orientation).
  • FIG. 4B NOEY2 -induced growth inhibition of ovarian and breast cancer cell lines. Inhibition of cyclin DI promoter activity in Saos-2, NIH3T3, SKBr3 and Hey cells. NOEY2 sense and antisense constructs were co-transfected with a luciferase reporter under the control of the human cyclin DI promoter (Albanese et al., 1995). Luciferase activity measured in cells transfected with the sense construct was expressed as a percentage of activity measured in cells transfected with the antisense. The results were from representative studies performed in triplicate.
  • FIG. 5A NBE 007 cell proliferation as measured by MTT (Ferrari et al, 1990). Primary cultures of NBE 007 were grown under two conditions: OSE medium or MEGM medium (Clonetics) which contains EGF, insulin, hydrocortisone and BPE.
  • FIG. 5B Expression of NOEY2 and p 21 WAF1/CIP1 in OSE and NBE cultures as assessed by Northern analysis.
  • FIG. 6A Induction of p21 WAF1/CIP1 expression by HA-NOEY2.
  • FIG. 6B Induction of p 21 WAF1 CIP1 expression by HA-NOEY2.
  • FIG. 6C HA-Erk2 transfectants.
  • P21 WAF1 CIP1 indicated in green (FITC).
  • FIG. 6D HA indicated in red (rhodamine).
  • FIG. 7. Map of the genomic structure of NOEY2. Exons 1 and 2 are show as well as the large intron 1. Nucleotide residue numbers are shown below.
  • FIG. 8. Expression in OSE.NBE and ovarian cancer cell lines (SKOv3 and Hey) of the wild type NOEY2 promoter linked to a luciferase reporter (p9) and the NOEY2 promoter with an A to G mutation at -750 (p9m 2-1.1 and p9m 4-1. An A to G mutation was introduced into the wild type NOEY2 promoter by site directed mutagenesis. The results are from representative studies performed in triplicate.
  • Germline mutation of several genes has recently been implicated in familial ovarian cancer including BRCAl (Miki et al., 1994), BRCA2 (Wooster et al, 1994) mismatch repair genes (Lynch et al, 1996) and, on rare occasions, p53 (Malkin et al., 1990).
  • BRCAl Mattle et al.
  • BRCA2 Wioster et al, 1994
  • p53 Malkin et al., 1990.
  • the inventors' laboratory has focused on defining the somatic changes that distinguish malignant from benign ovarian epithelium (Bast et al., 1992). With Larry Feig and Geoff Cooper, the inventors had found a mutant Ki-Ras gene in ovarian cancer cells, but not in benign mesothehal cells from the same patient's ascites (Feig et al.
  • Ras is constitutively activated in a significant fraction of ovarian cancer cell lines in the absence of mutation or amplification.
  • signaling can be documented through the MAP kinase pathway and the ets-2 transcription factor, possibly accounting for the larger fraction of ovarian cancers that overexpress c-myc and other Ras-responsive genes such as urokinase plasminogen activator and matrix metalloproteinases that are important for invasion.
  • Physiologic activation of Ras implies that receptors and/or transducing molecules upstream are activated.
  • a number of autocrine and paracrine growth regulatory pathways have been identified that appear to be altered in epithelial ovarian cancers when compared to normal ovarian epithelium.
  • the EGFR family (EGFR, HER-2, HER-3, HER-4) of tyrosine kinase growth factor receptors may be particularly important in ovarian cancer.
  • Normal ovarian surface epithelium expresses EGFR and can be stimulated to proliferate with exogenous EGF or TGF-(Rodriguez et al., 1991).
  • Ovarian carcinomas retain EGFR in 70% of cases (Berchuck et al, 1991) and can secrete EGF and TGF-.
  • Evidence for autocrine growth regulation has been obtained by inhibiting proliferation of ovarian cancer cell lines with anti-TGF- antibodies both in vitro and in vivo (Stromberg et al, 1992; Morishige et al, 1991).
  • the inventors have demonstrated that loss of EGFR expression by tumor cells is associated with a slight, but statistically significant improvement in survival (Berchuck et al, 1991), consistent with loss of autocrine growth stimulation.
  • Overexpression of HER-2 (c-erbB2) has been observed in 30% of cases of ovarian cancer.
  • HER-2 In stage III disease, overexpression of HER-2 is associated with a significantly shortened survival (Berchuck et al, 1990), although the significance of this marker in early stage disease is less certain (Rubin et al, 1993; Kacinski et al, 1992). Expression of HER-3 is increased in some borderline and early invasive ovarian tumors. Little has been reported regarding HER-4 in clinical material. Studies in cell culture suggest that the ligands heregulin and neu differentiating factor (NDF) signal through homodimers of HER-4 or heterodimers containing of HER-2/HER-3 or HER-2/HER-4. Cross-talk between the EGFR family members has been described.
  • NDF neu differentiating factor
  • HER- 2/HER-3 may be particularly important in determining whether growth of tumor cells is stimulated or inhibited by heregulin.
  • heregulin and NDF can inhibit, rather than stimulate clonogenic growth of ovarian and breast cancer cells that overexpress HER-2 in the presence of modest levels of HER-3 or HER-4 (Xu et al, 1996).
  • HER-2 tumor cells that overexpress HER-2 are growth inhibited by these ligands or by agonistic antibodies that bind only to HER-2
  • both ligands and antibodies can stimulate the ability of ovarian or breast cancer cells to invade matrigel membranes, associated with increased expression of matrix metalloproteinase 9 (MMP9) (Xu et al, 1994). Consequently, overexpression of HER-2 may potentiate the ability of tumor cells to invade and metastasize rather than to proliferate.
  • MMP9 matrix metalloproteinase 9
  • tyrosine kinase growth factor receptors have been identified in ovarian cancers that can signal through Ras and other pathways.
  • Normal ovarian surface epithelium expresses little, if any of the fins tyrosine kinase growth factor receptor and secretes low levels of its ligand macrophage colony stimulating factor (M-CSF or CSF-1) (Lidor et al, 1993).
  • M-CSF or CSF-1 macrophage colony stimulating factor
  • Approximately 50% of ovarian cancers express fins (Kacinski, 1995) and 70% secrete substantial levels of M-CSF (Xu et al, 1991), consistent with possible autocrine growth stimulation through the Ras pathway.
  • M-CSF is a potent chemoattractant for macrophages (Dorsch et al, 1993) which release a number of cytokines with growth regulatory activity for normal and transformed ovarian epithelium including tumor necrosis factor alpha (TNF- ⁇ ), IL-1 and IL-6 (Wu et al, 1992).
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-1 tumor necrosis factor alpha
  • Dr. Mills (Project 4) is studying OCAF, a novel lysophospholipid growth factor that is present in ascites fluid from a majority of ovarian cancer patients and stimulates tumor growth in more than 90% of cases. OCAF activates the MAP kinase pathway through a cascade involving Ras and tyrosine kinases.
  • Dr. Skinner (Project 1) is evaluating the role of several peptide factors including KGF, HGF and kit ligand that activate receptors which can also impinge on the Ras pathway.
  • TGF- 1 and 2 can be expressed by and activated in normal ovarian epithelial cells consistent with autocrine as well as paracrine growth inhibition (Berchuck et al, 1992).
  • Different ovarian cancer cell lines have lost the ability to express, activate or respond to TGF- (Berchuck et al. , 1992), but more than 90% ovarian cancer ascites tumor specimens can inhibited by TGF- and a fraction undergo apoptosis (Hurteau et al, 1994).
  • TGF- may provide a primitive surveillance mechanism for eliminating epithelial cells as they transform.
  • Expression of TGF- is lost in up to 40% of ovarian cancer specimens that presumably would have lost autocrine growth inhibition, but paracrine growth inhibition and induction of apoptosis could be obtained from TGF- secreted by the underlying stroma.
  • Other candidates for negative growth regulation of ovarian epithelial cells include the protein tyrosine phosphatases (PTPs) that can deactivate or reverse the effects of certain tyrosine kinases.
  • PTPs can, however, stimulate as well as inhibit growth of cells in different lineages.
  • the inventors' group has cloned fragments of 13 PTPs from ovarian cancers (Wiener et al, 1996).
  • PTP-1C and PTP-2A were regularly expressed in normal epithelial cells, but not expressed in a fraction of tumors, whereas PTP- IB, PTP- and PTP-H are upregulated in response to transfection of HER-2 (Wiener et al, 1996).
  • Evidence is mounting for the potential role of PTP-1C as a tumor suppressor in several different cell lineages (Shultz et al, 1993) and the inventors' own data indicate that expression of PTP-1C inhibits growth of ovarian cancer cells.
  • PTP- IB In clinical material a correlation has been observed between expression of PTP- IB and that of EGFR, HER-2 and fins (Wiener et ⁇ l, 1994). In experimental systems PTP- IB can suppress transformation induced by expression of mutant HER-2 (Brown-Shiner et ⁇ l, 1992). Overexpression of PTP- IB may reflect an inadequate homeostatic mechanism in ovarian carcinomas that have persistent or increased expression of tyrosine kinases such as EGFR, HER-2 and fins. Alternatively, the PTP- IB promoter may contain response elements for signaling pathways activated by tyrosine kinase receptors. 4.2 TUMOR SUPPRESSOR GENES IN EPITHELIAL OVARIAN CANCER
  • the tumor suppressor gene best studied in ovarian cancer is p53 which is mutated in approximately 50% of metastatic tumors, but in only 15% of lesions in stage IA or IB (Berchuck et ⁇ l., 1994). The gene is rarely affected in benign or borderline lesions.
  • the pattern of p53 mutation is most consistent with spontaneous deamination during normal replication rather than formation of adducts with exogenous carcinogens (Kohler et ⁇ l., 1993). Loss of heterozygosity at RB has been observed in a fraction of ovarian cancers, but functional RB protein is generally present.
  • Ras Ras oncogene proteins structurally related to the Ras oncogene proteins
  • the model of action of Ras has recently been intensively investigated, and one of its direct downstream target molecules has been identified to be c-raf-1, which induces the activation of the MAP kinase/ERK through MEK.
  • the Rap family consists of several highly homologous members - Rapl A, RaplB, and Rap2 - that belong to the Ras superfamily of small GTP -binding proteins (Pizon et al, 1988).
  • RaplA and/or RaplB have been shown to antagonize the Ras Functions, such as the Ki-Ras-induced transformation of NIH 3T3 cells (Kitayama et al, 1989), the Ha- Ras-induced germinal vesicle breakdown in Xenopus oocytes (Campa et al, 1991), the N-Ras- inhibited muscarinic K + channel activity (Yatani et al, 1991), the Ki-Ras-induced activation of the c-fos promoter/enhancer in NIH 3T3 cells (Sakoda et al, 1992), the proliferation of middle T antigen-transformed Rat-2 cells (Jelinek and Hassell, 1992), and the Ha-Ras-induced activation of the c-Raf-1 protein kinase-dependent Map kinase cascade in Rat-1 cells (Cook et al, 1993).
  • the Ki-Ras-induced transformation of NIH 3T3 cells
  • RaplA and/or RaplB have been shown to be phosphorylated by protein kinase A in both intact cells and cell-free systems (Quilliam et al, 1991), by Ca2 + /calmodulin-dependent protein kinase Gr in a cell-free system (Sahyoun et al, 1991), and by protein kinase G in a cell- free system (Miura et al, 1992).
  • the protein kinase A-catalyzed phosphorylation sites of RaplA and RaplB are Ser 180 and Ser 179, respectively, in their C-terminal regions (Quilliam et al, 1991; Hata et al, 1991).
  • RaplB This phosphorylation of RaplB lowers its membrane binding activity and induces its translocation from the membrane to the cytosol (Hata et al, 1991).
  • the phosphorylation of RaplB makes it sensitive to the action of Smg GDS to stimulate its GDP/GTP exchange reaction (Hata et al, 1991).
  • the GTP binding site of Ras proteins consists of four non-contiguous regions encountered in all the proteins of the Ras superfamily. Among these regions six amino acids: DTAGQE, in positions 57 to 62 of the K-Ras protein, seemed to be a hallmark of all the Ras and Ras-related proteins. It is known that single amino acid substitution in p21Ras at aal2 (glycine), 13 (glycine) and 61 (glutamine) significantly reduce the intrinsic GTPase activity of Ras proteins and prevent Ras-GAP from accelerating the rate of GTP hydrolysis. Therefore, it appears that this domain plays an essential role in the control of the biological properties of the Ras proteins. Random mutagenesis studies also shown that amino acid substitutions at positions 59 and 63 can activate Ras transforming potential. 4.4 Loss OF HETEROZYGOSITY IN OVARIAN CANCERS
  • LOH has been observed within cancers at the site of deleted tumor suppressor genes. Numerous studies of loss of heterozygosity (LOH) in ovarian carcinoma have been published since 1989 and are all largely based on techniques using normal-tumor pairs. Sites of LOH have been reported on lp, 3p, 4p, 6p, 7p, 8q, l ip, 13q, 17p, 17q, 18q and 22. Studies have detected LOH at sites distal to BRCAl on chromosome 17q (Hacibs et al, 1993) and at the BRCA2 site on chromosome 13q. The short arm of chromosome 1 is frequently affected by rearrangements in a variety of human malignancies.
  • polypeptides may be desirable to purify NOEY2 polypeptides, NOEY2 epitopes, NOEY2-derived peptide fragments, or variants thereof.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%o, about 80%), about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High performance liquid chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of min, or at most an h. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • Gel chromatography is uns ⁇ jpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adso ⁇ tion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.
  • Lectins are usually coupled to agarose by cyanogen bromide.
  • Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl ghicosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands.
  • Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D-galactosamine is used for purifying lectins from soybean; N-acetylglucosamine binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffmity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • NOEY2-derived peptides and polypeptide fragments for use in various diagnostic and therapeutic applications.
  • the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1966); Voss et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each inco ⁇ orated herein by reference.
  • Short peptide sequences or libraries of overlapping peptides, usually from about 6 or so amino acids, and up to and including about 35 to 50 or so amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • the present invention also provides for the use of NOEY2 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either NOEY2, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • mutant tumor suppressors may not be non-functional. Rather, they may have aberrant functions that cannot be overcome by replacement gene therapy, even where the "wild-type" molecule is expressed in amounts in excess of the mutant polypeptide. Therefore, an important aspect of the invention concerns the preparation and use of NOEY2 antisense constructs. Such antisense technology may be used to "knock-out” or reduce the function or expression of NOEY2 in a cell, or may ablate the function of NOEY2 in the development of cell line or in a transgenic mouse or other animal used in research, or diagnostic and/or screening methods.
  • antisense techniques take advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology as well as non-homologous regions (e.g., ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions. The preparation and use of such ribozymes are described in detail in the following section.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U. S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence- specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
  • RNA Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
  • the enzymatic nucleic acid molecule may be formed in a hammerhead, hai ⁇ in, a hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif.
  • hammerhead motifs are described by Rossi et al. (1992).
  • hai ⁇ in motifs are described by Hampel et al (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and U. S. Patent 5,631,359 (specifically inco ⁇ orated herein by reference).
  • hepatitis ⁇ virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991 ; Collins and Olive, 1993); and an example of the Group I intron is described in (U. S. Patent 4,987,071, specifically inco ⁇ orated herein by reference).
  • an enzymatic nucleic acid molecule of this invention has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
  • the ribozyme constructs need not be limited to specific motifs mentioned herein.
  • the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA.
  • Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required.
  • the ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.
  • Small enzymatic nucleic acid motifs may also be used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure.
  • catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon et al, 1991; Kashani-Sabet et al, 1992; Dropulic et al, 1992; Weerasinghe et al, 1991; Ojwang et al, 1992; Chen et al, 1992; Sarver et al, 1990).
  • any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector.
  • the activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl. Publ. No. WO 94/02595, both hereby inco ⁇ orated by reference; Ohkawa et al, 1992; Taira et al, 1991; and Ventura et al, 1993).
  • Ribozymes may be added directly, or can be complexed with cationic lipids, lipid complexes, packaged within liposomes, or otherwise delivered to target cells.
  • the RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their inco ⁇ oration in biopolymers.
  • Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically inco ⁇ orated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.
  • Hammerhead or hai ⁇ in ribozymes may be individually analyzed by computer folding (Jaeger et ⁇ l, 1989) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 or so bases on each arm are able to bind to, or otherwise interact with, the target RNA.
  • Ribozymes of the hammerhead or hai ⁇ in motif may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized.
  • the method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et ⁇ l. (1987) and in Scaringe et ⁇ l. (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Average stepwise coupling yields are typically >98%.
  • Hai ⁇ in ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).
  • Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992).
  • Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.
  • Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et ⁇ l, 1990; Pieken et ⁇ l, 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Patent 5,334,711; and Int. Pat.
  • Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by inco ⁇ oration into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically inco ⁇ orated herein by reference.
  • RNA polymerase I RNA polymerase I
  • RNA polymerase II RNA polymerase II
  • RNA polymerase III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al, 1993; Zhou et al, 1990). Ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani- Saber et al, 1992; Ojwang et al, 1992; Chen et al, 1992; Yu et al, 1993; L ⁇ uillier et al, 1992; Lisziewicz et al, 1993).
  • transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, Sindbis virus vectors).
  • plasmid DNA vectors such as adenovirus or adeno-associated vectors
  • viral RNA vectors such as retroviral, semliki forest virus, Sindbis virus vectors.
  • Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. They can also be used to assess levels of the target RNA molecule. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base- pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease.
  • ribozymes of this invention include detection of the presence of mRNA associated with an IL-5 related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.
  • expression vectors are employed to express a NOEY2 or NOEY2-derived polypeptide product, which can then be purified and, for example, be used to vaccinate animals, or to generate antisera or monoclonal antibodies which may be used in a variety of diagnostic and therapeutic applications.
  • an expression vector comprising a NOEY2 or NOEY2-derived polynucleotide may be used in gene therapy.
  • Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatabihty in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide. 4.10.1 REGULATORY ELEMENTS
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • a sequence encodes all or part of a gene which encodes a NOEY2 polypeptide.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product.
  • expression only includes transcription of the nucleic acid encoding a gene of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • a host cell transformed with one or more NOEY2 nucleic acid segments may be identified in vitro or in vivo by including a "marker” or “reporter” gene in the expression construct and/or vector which comprises the NOEY2 polynucleotide.
  • reporter or marker would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants.
  • Genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are often employed as selectable markers.
  • enzymes such as he ⁇ es simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may also be employed, as well as one or more immunologic markers.
  • selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • Knowledge of the genetic organization of adenovirus a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.
  • Adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity.
  • Generation and propagation of adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977).
  • the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991).
  • adenovirus can package approximately 105%) of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector.
  • MOI multiplicities of infection
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the preferred helper cell line is 293.
  • the medium is then replaced with 50 ml of fresh medium and shaking initiated.
  • cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 ⁇ plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et ⁇ l., 1963; Top et ⁇ l., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et ⁇ l, 1991; Gomez-Foix et ⁇ l, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et ⁇ l, 1990; Rich et ⁇ l., 1993).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981).
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and he ⁇ esviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • the expression construct In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
  • nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes.
  • DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be inco ⁇ orated by the present invention.
  • such expression constructs may be entrapped in a liposome, lipid complex, nanocapsule, or other formulation using one or more of the methods disclosed in Section 4.8.
  • lipofectamine-DNA complexes are also contemplated.
  • liposome- mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Nicolau et al (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • bacterial promoter is employed in the DNA construct
  • Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer.
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor.
  • Mannose can be used to target the mannose receptor on liver cells.
  • antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
  • One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins.
  • the gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions.
  • the gene for virtually any polypeptide may be employed in this manner.
  • the generation of recombinant expression vectors, and the elements included therein, are discussed above.
  • the protein to be produced may be an endogenous protein normally synthesized by the cell in question. Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
  • a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti- metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e. a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
  • the main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1.
  • the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
  • liposomes and/or nanocapsules for the introduction of a NOEY2 composition into a host cell.
  • Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the polypeptides, pharmaceuticals, and/or antibodies disclosed herein.
  • the formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al , 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). More recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).
  • the disclosed composition may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
  • the term "liposome” is intended to mean a composition arising spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Nanocapsules can generally entrap compounds in a stable and reproducible way
  • compositions containing nanocapsules for the oral delivery of active agents are described in U.S. Patent 5,500,224 and U.S. Patent 5,620,708.
  • U.S. Patent 5,500,224 describes a pharmaceutical composition in the form of a colloidal suspension of nanocapsules comprising an oily phase consisting essentially of an oil containing dissolved therein a surfactant and suspended therein a plurality of nanocapsules having a diameter of less than 500 nanometers.
  • U.S. Patent 5,620,708 describes compositions and methods for the oral administration of drugs and other active agents.
  • the compositions comprise an active agent carrier particle attached to a binding moiety which binds specifically to a target molecule present on the surface of a mammalian enterocyte.
  • the binding moiety binds to the target molecule with a binding affinity or avidity sufficient to initiate endocytosis or phagocytosis of the particulate active agent carrier so that the carrier will be absorbed by the enterocyte.
  • the active agent will then be released from the carrier to the host's systemic circulation.
  • degradation-sensitive drugs such as polypeptides
  • U.S. Patent 5,641,515 and U.S. Patent 5,698,515 describe the use of nanocapsules for the oral administration of a polypeptide, specifically, insulin and are inco ⁇ orated herein by reference.
  • Patent 5,698,515 described insulin containing nanocapsules intended for oral administration of insulin which comprises a hydrophilic polymer modified with an inhibitor of proteolytic enzyme, insulin and water, wherein the inhibitor of proteolytic enzymes is ovomucoid isolated from duck or turkey egg whites.
  • US. Patent 5,556,617 describes the use of nanoparticles as pharmaceutical treatment of the upper epidermal layers by topical application on the skin.
  • Poly(alkyl cyanoacrylate) nanocapsules have been used as biodegradable polymeric drug carriers for subcutaneous and peroral delivery of octreotide, a long-acting somatostatin analogue.
  • the nanocapsules prepared by interfacial emulsion polymerization of isobutyl cyanoacrylate, were 216 nm in diameter and inco ⁇ orated 60% of octreotide. Nanocapsules were administered subcutaneously and the octreotide-loaded nanocapsules (20 mg/kg) suppressed the insulinaemia peak induced by intravenous glucose overload and depressed insulin secretion over 48 h.
  • octreotide loaded nanocapsules significantly improved the reduction of prolactin secretion and slightly increased plasma octreotide levels (Damge et al, 1997).
  • nanocapsules make them particularly susceptible to lysozyme (LZM), a positively-charged enzyme that is highly concentrated in mucosas. This interaction causes destabilization of the nanocapsule by LZM; however, it was observed that the destabilizing effects caused by the adso ⁇ tion of LZM onto the nanocapsules can be prevented by previous adso ⁇ tion of the cationic poly(amino acid) poly-L-lysine (Calvo et al, 1997).
  • LZM lysozyme
  • PECL poly-epsilon-caprolactone
  • the idea is based on a graft copolymer model embodying a link site for attachment to the carrier, a floating pad for maintaining the particles afloat in the blood stream, an affinity ligand for site-specific delivery and a structural tune for balancing the overall structure of the homing device.
  • U. S. Patent 5,451,410 describes the use of modified amino acid for the encapsulation of active agents.
  • Modified amino acids and methods for the preparation and used as oral delivery systems for pharmaceutical agents are described.
  • the modified amino acids are preparable by reacting single amino acids or mixtures of two or more kinds of amino acids with an amino modifying agent such as benzene sulfonyl chloride, benzoyl chloride, and hippuryl chloride.
  • the modified amino acids form encapsulating microspheres in the presence of the active agent under sphere-forming conditions.
  • the modified amino acids may be used as a carrier by simply mixing the amino acids with the active agent.
  • the modified amino acids are particularly useful in delivering peptides, e.g., insulin or calmodulin, or other agents which are sensitive to the denaturing conditions of the gastrointestinal tract.
  • NOEY2 4.12 DIAGNOSING CANCERS INVOLVING NOEY2
  • a NOEY2 polypeptide or a NOEY2 gene may be employed as a diagnostic or prognostic indicator of cancer. More specifically, point mutations, deletions, insertions or regulatory perturbations relating to NOEY2 may cause cancer or promote cancer development, cause or promoter tumor progression at a primary site, and/or cause or promote metastasis. Other phenomena associated with malignancy that may be affected by NOEY2 expression include angiogenesis and tissue invasion.
  • One embodiment of the instant invention comprises a method for detecting variation in the expression of NOEY2.
  • This may comprises determining that level of NOEY2 or determining specific alterations in the expressed product.
  • this sort of assay has importance in the diagnosis of related cancers.
  • Such cancer may involve cancers of the breast or ovaries, or alternatively, cancers involving the lung, liver, spleen, brain kidney, pancreas, small intestine, blood cells, lymph node, colon, endometrium, stomach, prostate, testicle, skin, head and neck, esophagus, bone marrow, blood or other tissue.
  • the present invention relates to the diagnosis of breast and ovarian cancers.
  • the biological sample can be any tissue or fluid.
  • Various embodiments include cells of the skin, muscle, facia, brain, prostate, breast, endometrium, lung, head & neck, pancreas, small intestine, blood cells, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow or kidney.
  • Other embodiments include fluid samples such as peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.
  • Nucleic acid used is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA.
  • RNA may be desired to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA; in another, it is poly-A RNA.
  • the nucleic acid is amplified.
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • alterations should be read as including deletions, insertions, point mutations and duplications. Point mutations result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those occurring in non-germline tissues. Germ-line tissue can occur in any tissue and are inherited. Mutations in and outside the coding region also may affect the amount of NOEY2 produced, both by altering the transcription of the gene or in destabilizing or otherwise altering the processing of either the transcript (mRNA) or protein.
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • SSCA single-stranded conformation analysis
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCR-SSCP.
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from about ten to about fifteen base pairs in length or even longer sequences such as those from about twenty to about 30 base pairs or more in length, with even longer sequences be employed for certain applications.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred. Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species ( P, C, S, H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemiluminescent (luciferase).
  • radioactive species P, C, S, H, or other label
  • fluorophore rhodamine, fluorescein
  • luciferase chemiluminescent
  • PCRTM polymerase chain reaction
  • PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • RT-PCRTM reverse transcriptase PCRTM amplification procedure
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • PCT/US87/00880 may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]- triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992).
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Still another amplification methods described in GB Application No. 2,202,328, and in
  • PCT Application No. PCT/US89/01025 each of which is inco ⁇ orated herein by reference in its entirety, may be used in accordance with the present invention.
  • modified primers are used in a PCR-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Int. Pat. Appl. Publ. No. WO 88/10315, inco ⁇ orated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989; Int. Pat. Appl. Publ. No. WO 88/10315, inco ⁇ orated herein by reference in their entirety.
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, 1990; Ohara et al, 1989; each herein inco ⁇ orated by reference in their entirety).
  • Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose.
  • a suitable matrix often a filter of nitrocellulose.
  • the different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • a probe usually labeled
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.
  • chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder et ⁇ /., 1968a, Freifelder et al. , 1968b; Freifelder, 1982).
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products are integrally labeled with radio- or fiuorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by a labeled probe.
  • the techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols (see Sambrook et al, 1989).
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • U. S. Patent No. 5,279,721, inco ⁇ orated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout the NOEY2 gene that may then be analyzed by direct sequencing.
  • kits This generally will comprise preselected primers and probes. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • RT polymerases
  • Taq Taq
  • SequenaseTM a polymerases
  • buffers to provide the necessary reaction mixture for amplification.
  • kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • RT Reverse transcription
  • RT-PCRTM relative quantitative PCRTM
  • PCRTM the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles.
  • a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCRTM amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCRTM reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCRTM products and the relative mRNA abundance is only true in the linear range of the PCRTM reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundance of a mRNA species can be determined by RT-PCRTM for a collection of RNA populations is that the concentrations of the amplified PCRTM products must be sampled when the PCRTM reactions are in the linear portion of their curves.
  • the second condition that must be met for an RT-PCRTM experiment to successfully determine the relative abundance of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • the goal of an RT-PCRTM experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • mRNAs for ⁇ -actin, asparagine synthetase and lipocortin II were used as external and internal standards to which the relative abundance of other mRNAs are compared.
  • RT-PCRTM assay for clinically derived materials.
  • the problems inherent in clinical samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the RT-PCRTM is performed as a relative quantitative RT-PCRTM with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • RT-PCRTM assays can be superior to those derived from the relative quantitative RT- PCRTM assay with an internal standard.
  • One reason for this advantage is that without the internal standard/competitor, all of the reagents can be converted into a single PCRTM product in the linear range of the amplification curve, thus increasing the sensitivity of the assay.
  • Another reason is that with only one PCRTM product, display of the product on an electrophoretic gel or another display method becomes less complex, has less background and is easier to inte ⁇ ret.
  • chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991).
  • NOEY2 nucleic acids in the screening of compounds for activity in either stimulating NOEY2 activity, overcoming the lack of NOEY2, or blocking the effect of a mutant NOEY2 molecule.
  • assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.
  • Contemplated functional "read-outs” include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, phosphatase activity, anti-phosphatase activity, phosphorylation of NOEY2, dephosphorylation of NOEY2, inhibition or stimulation of cell-to- cell signaling, growth, metastasis, cell division, cell migration, soft agar colony formation, contact inhibition, invasiveness, angiogenesis, apoptosis, tumor progression or other malignant phenotype.
  • the invention is to be applied for the screening of compounds that bind to the NOEY2 molecule or fragment thereof.
  • the polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting determining of binding.
  • the assay may measure the inhibition of binding of NOEY2 to a natural or artificial substrate or binding partner.
  • Competitive binding assays can be performed in which one of the agents (NOEY2, binding partner or compound) is labeled.
  • the polypeptide will be the labeled species.
  • One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
  • WO 84/0356 Another technique for high throughput screening of compounds is described in WO 84/03564.
  • Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with NOEY2 and washed. Bound polypeptide is detected by various methods.
  • Purified NOEY2 can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
  • fusion proteins containing a reactive region may be used to link the NOEY2 active region to a solid phase.
  • Various cell lines containing wild-type or natural or engineered mutations in NOEY2 can be used to study various functional attributes of NOEY2 and how a candidate compound affects these attributes.
  • Methods for engineering mutations are described elsewhere in this document, as are naturally-occurring mutations in NOEY2 that lead to, contribute to and/or otherwise cause malignancy.
  • the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell.
  • culture may be required.
  • the cell may then be examined by virtue of a number of different physiologic assays.
  • molecular analysis may be performed in which the function of NOEY2, or related pathways, may be explored. This may involve assays such as those for protein expression, enzyme function, substrate utilization, phosphorylation states of various molecules including NOEY2, cAMP levels, mRNA expression (including differential display of whole cell or polyA RNA) and others.
  • the present invention also encompasses the use of various animal models.
  • the identity seen between human and mouse NOEY2 provides an excellent opportunity to examine the function of NOEY2 in a whole animal system where it is normally expressed.
  • By developing or isolating mutant cells lines that fail to express normal NOEY2 one can generate cancer models in mice that will be highly predictive of cancers in humans and other mammals.
  • These models may employ the orthotopic or systemic administration of tumor cells to mimic primary and/or metastatic cancers.
  • one may induce cancers in animals by providing agents known to be responsible for certain events associated with malignant transformation and/or tumor progression.
  • transgenic animals discussed below
  • that lack a wild-type NOEY2 may be utilized as models for cancer development and treatment.
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route the could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • systemic intravenous injection regional administration via blood or lymph supply and intratumoral injection.
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, survival, reduction of tumor burden or mass, arrest or slowing of tumor progression, elimination of tumors, inhibition or prevention of metastasis, increased activity level, improvement in immune effector function and improved food intake.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • An alternative approach, "alanine scan” involves the random replacement of residues throughout molecule with alanine, and the resulting affect on function determined.
  • NOEY2-specific antibody selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • drugs which have improved NOEY2 activity or which act as stimulators, inhibitors, agonists, antagonists or NOEY2 or molecules affected by NOEY2 function.
  • sufficient amounts of NOEY2 can be produced to perform crystallographic studies.
  • knowledge of the polypeptide sequences permits computer employed predictions of structure- function relationships.
  • compositions comprising e.g., expression vectors, virus stocks, polypeptides, polynucleotides, antibodies and/or drugs
  • pharmaceutical compositions comprising e.g., expression vectors, virus stocks, polypeptides, polynucleotides, antibodies and/or drugs
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be inco ⁇ orated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • a transgenic animal is produced which contains one or more functional transgenes that encode a functional NOEY2 polypeptide or a variant thereof.
  • Transgenic animals expressing NOEY2 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of NOEY2.
  • Transgenic animals of the present invention may also be used as models for studying indications such as cancers.
  • a NOEY2 transgene is introduced into a non- human host to produce a transgenic animal expressing a NOEY2 gene.
  • the transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene.
  • Methods for producing transgenic animals are generally described by Wagner and Hoppe (U. S. Patent No. 4,873,191; which is inco ⁇ orated herein by reference), Brinster et al.
  • mice rats, monkeys, hamsters, pigs, dogs, cats, goats, rabbits, horses, sheep, or virtually any other animal for which methods have been developed for introducing a stable transgene into its germline.
  • NOEY2 gene(s) may be replaced by homologous recombination between the transgene and the endogenous gene; or the endogenous gene may be eliminated by deletion as in the preparation of "knock-out" animals.
  • a NOEY2 gene flanked by genomic sequences is transferred by microinjection into a fertilized egg.
  • the microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene.
  • Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
  • transgenic mice are generated which overexpress NOEY2 or express a mutant form of the polypeptide.
  • NOEY2 overexpress NOEY2 or express a mutant form of the polypeptide.
  • the absence of NOEY2 in "knock-out" mice permits the study of the effects that loss of NOEY2 protein has on a cell in vivo.
  • Knock-out mice also provide a model for the development of NOEY2 -related cancers, and particularly ovarian and breast cancers.
  • transgenic animals and cell lines derived from such animals may find use in certain testing studies.
  • transgenic animals and cell lines capable of expressing wild-type or mutant NOEY2 may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type NOEY2 expression and or function or impair the expression or function of mutant NOEY2.
  • Mutagenesis may be performed in accordance with any of the techniques known in the art such as and not limited to synthesizing an oligonucleotide having one or more mutations within the sequence of a particular promoter region.
  • site-specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.
  • site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site- directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired promoter region or peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • a genetic selection scheme was devised by Kunkel et al. (1987) to enrich for clones inco ⁇ orating the mutagenic oligonucleotide.
  • the use of PCRTM with commercially available thermostable enzymes such as Taq polymerase may be used to inco ⁇ orate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector.
  • a PCRTM employing a thermostable ligase in addition to a thermostable polymerase may also be used to inco ⁇ orate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector.
  • the mutagenesis procedure described by Michael (1994) provides an example of one such protocol.
  • sequence variants of the selected promoter-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of DNA sequences may be obtained.
  • recombinant vectors encoding the desired promoter sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification.
  • oligonucleotide directed mutagenesis procedure also is intended to refer to a process that involves the template-dependent extension of a primer molecule.
  • template- dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically inco ⁇ orated herein by reference in its entirety.
  • a number of template dependent processes are available to amplify the target sequences of interest present in a sample.
  • One of the best known amplification methods is the polymerase chain reaction (PCRTM) which is described in detail in U. S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is inco ⁇ orated herein by reference in its entirety.
  • PCRTM two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase).
  • a DNA polymerase e.g., Taq polymerase
  • the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880, inco ⁇ orated herein by reference in its entirety, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which can then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]triphosphates in one strand of a restriction site (Walker et al, 1992, inco ⁇ orated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, e.g., nick translation.
  • a similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
  • RCR Repair Chain Reaction
  • a similar approach is used in SDA.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al, 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, inco ⁇ orated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has crystal protein-specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second crystal protein- specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate crystal protein-specific sequences.
  • a polymerase such as T7 or SP6
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification of DNA sequences of the present invention.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 1.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, inco ⁇ orate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (- 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • Patent 4,554,101 inco ⁇ orated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate
  • leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present invention also involves, in another embodiment, the treatment of cancer.
  • the types of cancer that may be treated, according to the present invention is limited only by the involvement of NOEY2. By involvement, it is not even a requirement that NOEY2 be mutated or abnormal - the overexpression of this tumor suppressor may actually overcome other lesions within the cell.
  • NOEY2 a wide variety of tumors may be treated using NOEY2 therapy, particularly those of the breast and ovaries, but also cancers of the lung, liver, spleen, brain kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, endometrium, prostate, testicle, skin, head and neck, esophagus, bone marrow, blood or other tissue.
  • the tumor cell be killed or induced to undergo normal cell death or "apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.
  • One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in the tumorigenesis of some cancers.
  • the present inventors intend to provide, to a cancer cell, an expression construct capable of providing NOEY2 to that cell.
  • NOEY2 transcripts have been identified not only in humans, but also in rat, mouse, monkey, and hamster, any of these nucleic acids could be used in human or animal therapy, as could any of the gene sequence variants discussed above which would encode the same, or a biologically equivalent polypeptide.
  • the development and use of such genes for treatment of cancers using a "gene therapy" approach are well known to those of skill in the art.
  • viral vectors such as adenovirus, adeno-associated virus, he ⁇ esvirus, vaccinia virus and retrovirus.
  • an expression vector that is contained within, or formulating using encapsulation within a lipid vesicle, lipid particle, liposome, or liposome-derived composition.
  • Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations.
  • viral vectors For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one may deliver anywhere on the order of from about 1 10 to about 1 x10 infectious particles to the patient. Alternatively, one may deliver higher concentrations of infectious particles to the patient, on the order of from about 1 xlO 9 to about 1 x10 or higher, depending upon the particular formulation, application, or cancer to be treated. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Formulation as a pharmaceutically acceptable composition is known in the art, as discussed supra.
  • routes are contemplated for various tumor types. The section below on routes contains an extensive list of possible routes. For practically any tumor, systemic delivery is contemplated. This will prove especially important for attacking microscopic or metastatic cancer.
  • discrete tumor mass may be identified, a variety of direct, local and regional approaches may be taken.
  • the tumor may be directly injected with the expression vector.
  • a tumor bed may be treated prior to, during or after resection. Following resection, one generally will deliver the vector by a catheter left in place following surgery.
  • One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery.
  • a more distal blood supply route also may be utilized.
  • ex vivo gene therapy is contemplated. This approach is particularly suited, although not limited, to treatment of bone marrow associated cancers.
  • cells from the patient are removed and maintained outside the body for at least some period of time. During this period, a therapy is delivered, after which the cells are reintroduced into the patient; hopefully, any tumor cells in the sample have been killed.
  • ABMT Autologous bone marrow transplant
  • the notion behind ABMT is that the patient will serve as his or her own bone marrow donor.
  • a normally lethal dose of irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo.
  • NOEY2 Use of gene therapy to accomplish this goal is yet another way NOEY2 may be utilized according to the present invention.
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • NOEY2 could serve as a target for an immune effector given that (a) it is unlikely to be expressed on the surface of the cell and (b) that the presence, not absence, of NOEY2 is associated with the normal state.
  • particular mutant forms of NOEY2 may be targeted by immunotherapy, either using antibodies, antibody conjugates or immune effector cells.
  • immunotherapy could be used as part of a combined therapy, in conjunction with NOEY2-targeted gene therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • tumor marker exist and any of these may be suitable for targeting in the context of the present invention.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • NOEY2 replacement therapy could be used similarly in conjunction with chemo- or radiotherapeutic intervention. It also may prove effective to combine NOEY2 gene therapy with immunotherapy, as described above.
  • To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present invention, one would generally contact a "target" cell with a NOEY2 expression construct and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same time.
  • This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
  • the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from min to wk.
  • the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell.
  • Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell.
  • Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV- irradiation, microwaves, electronic emissions, and the like.
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP- 16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • the use of cisplatin in combination with a NOEY2 expression construct is particularly preferred as this compound.
  • the tumor cells In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves.
  • the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a NOEY2 expression construct, as described above.
  • Agents that directly cross-link nucleic acids, specifically DNA, are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with NOEY2.
  • Agents such as cisplatin, and other DNA alkylating agents may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m for 5 days every three wk for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via inj ection intravenously, subcutaneously, intratumorally or intraperitoneally .
  • Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m at 21 day intervals for adriamycin, to 35-50 mg/m for etoposide intravenously or double the intravenous dose orally.
  • nucleic acid precursors and subunits also lead to DNA damage.
  • nucleic acid precursors have been developed.
  • agents that have undergone extensive testing and are readily available are particularly useful.
  • agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
  • 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
  • DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves and UV- irradiation. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. The skilled artisan is directed to "Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the chemo- or radiotherapy may be directed to a particular, affected region of the subjects body.
  • systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
  • NOEY2-targeted therapies with chemo- and radiotherapies
  • combination with other gene therapies will be advantageous.
  • targeting of NOEY2 and p53 or pi 6 mutations at the same time may produce an improved anti-cancer treatment.
  • any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, pi 6, FHIT, WT-1, MEN-I, MEN-II, BRCAl, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl. It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating a NOEY2-related cancer. In this regard, reference to chemotherapeutics and non-NOE72 gene therapy in combination should also be read as a contemplation that these approaches may be employed separately.
  • ELISAs may be used in conjunction with the invention.
  • proteins or peptides inco ⁇ orating tumor suppressor protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder.
  • BSA bovine serum albumin
  • casein casein
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background.
  • the layered antisera is then allowed to incubate for from about 2 to about 4 h, at temperatures preferably on the order of about 25° to about 27°C.
  • the antisera-contacted surface is washed so as to remove non-immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 h at room temperature in a PBS -containing solution such as PBS Tween®).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2, 2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2, 2'-azino-di-(3-ethyl- benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • the anti-tumor suppressor protein antibodies of the present invention
  • Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
  • For the isolation of membrane proteins cells must be solubilized into detergent micelles.
  • Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. enzyme-substrate pairs. 4.20 WESTERN BLOTS
  • the NOEY2 compositions of the present invention will find great use in immunoblot or western blot analysis.
  • the anti-NOEY2 antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immuno-precipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
  • Expression The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.
  • Promoter A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
  • Structural gene A gene that is expressed to produce a polypeptide. Transformation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is inco ⁇ orated into a chromosome or is capable of autonomous replication.
  • exogenous DNA sequence e.g., a vector, a recombinant DNA molecule
  • Transformed cell A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.
  • Transgenic cell Any cell derived or regenerated from a transformed cell or derived from a transgenic cell.
  • Vector A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment.
  • a plasmid is an exemplary vector.
  • EXAMPLE 1 - ISOLATION AND CHARACTERIZATION OF THE NOEY2 GENE The inventors have utilized differential display of mRNA by means of the polymerase chain reaction (DDPCRTM) (Liang and Pardee, 1992) to isolate a novel gene-NOEY2 from normal ovarian epithelial cells, it may serve as a tumor suppresser gene in ovarian and breast cancer.
  • DDPCRTM polymerase chain reaction
  • OSE ovarian surface epithelial
  • the normal OSE cells culture are obtained by gently scraping the surface of ovaries from patients undergoing surgery for nonmalignant gynecological diseases.
  • OSE cells can only be maintained in culture for 8 to 10 passages.
  • the cell cycle studies showed that most of OSE cells stably arrest growth with a GI DNA content, only small part of OSE cells had the ability to enter S phase. Since epithelial cystoadenocarcinoma constitute the large majority of ovarian malignancies, OSE cells provide a good model to investigate this cancer.
  • DDPCRTM Differential Display Polymerase Chain Reaction
  • the inventors have isolated and cloned a novel gene - NOEY2 - it is expressed in normal ovarian epithelial cells but consistently absent or down-regulated in the ovarian cancer cell lines.
  • RACE rapid amplification of cDNA ends
  • the first strand cDNA is synthesized from ovarian epithelial cell RNA using an NOEY2-specific primer SP1, AMV reverse transcriptase and the deoxynucleotide mixture.
  • terminal transferase is used to add a homopolymeric A-tail to the 3' end of the cDNA.
  • Tailed cDNA is then amplified by PCRTM using a NOEY2 specific primer SP2 and the oligo dT-anchor primer.
  • SP2 a NOEY2 specific primer
  • the oligo dT-anchor primer As a result the 5' RACE products were cloned into the TA vector for subsequent characterization, which included sequencing and restriction mapping.
  • a cDNA library from OSE cells constructed in Lambda ZAPII were screened with the extended NOEY2 cDNA. 5 ⁇ g of mRNA was purified from OSE cells.
  • the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA) was used to construct OSE cDNA library.
  • This cDNA library provided a method by which the transcription and processing of ovarian epithelial cell mRNA can be examined.
  • the extended NOEY2 cDNA from RACE (Boehringer Mannheim, Indianapolis, IN) was P-dCTP labeled by a random primer and used for screening OSE cDNA library. This screening procedure is performed on bacteriophage plaques, the OSE cDNA library is spread out on agarose plates, then the clones were transferred to filter membranes. The clones were hybridized to NOEY2 DNA probe. The positive clones were subsequently analyzed, they encoded the mRNA sequence and allowed prediction of the amino acid sequence.
  • FIG. 1A and FIG. IB close to a full-length NOEY2 cDNA sequence and open reading from (ORF) was obtained (FIG. 1A and FIG. IB).
  • the nucleotide sequence (SEQ ID NO:l) contains a 5' untranslated region of 149 bp, an open reading frame of 684 bp encoding a protein of 228 amino acids (SEQ ID NO:2) ending with a TGA codon and 646 bp of 3' untranslated sequence.
  • Translation of the NOEY2 protein yielded a protein of approximately 26 kDa.
  • nucleotide and amino acid sequence of the ORF was used to search the Genbank EST database using Netscape search GenBank of National Center for Biotechnology Information (NCBI) in EST database level I and level II. The most significant similarity detected was with members of the Ras and Rap gene family. NOEY2 shares 58%> amino acid homology with RaplA, 56% with RaplB, 58% with Rap 2, 61% with Rap2B, 51% with c-K-Ras and 54% with H-Ras.
  • the NOEY2 gene ORF exhibits three features similar to those of Ras/Rap family members: (a) a highly conserved GTP binding domain, (b) a putative effector domain YLPTIENTY (SEQ ID NO:3), and (c) the membrane localizing CAAX motif (where C is cysteine, A is an aliphatic amino acid and X is any amino acid) at the COOH terminus (FIG. 2).
  • NOEY2 differs both from Ras and Rap family members where the sequence YDPTIEDSY (SEQ ID NO:4) is found in all Ras and Rap genes. NOEY2 instead has YLPTIENTY. NOEY2 sequence had substitutions of alanine for glycine at amino acid 12 and valine for glutamine at amino acid 61 when compared to p21 ras , consistent with constitutive activation of the G protein if it behaved in a manner similar to Ras.
  • RNA samples (15 ⁇ g) were size-fractionated by formaldehyde/agarose gel electrophoresis and transferred to a hybond-nylon membrane. The membranes were UV-cross liked, prehybridized
  • NOEY2 was expressed in all eleven primary normal OSE cultures and all ten primary normal breast epithelial cultures, but was lost in 11 of 12 ovarian cancer cell lines (FIG. 3 A) and 9 of 9 breast cancer cell lines (FIG. 3B).
  • NOEY2 expression was also lost in each of 9 primary ovarian cancer cell preparations that were separated and purified from patients' ascites (FIG. 3C).
  • FIG. 3C Using multiple tissue blots containing polyA RNA from sixteen different normal human tissues, the NOEY2 gene was found in several other normal tissues, including heart, liver, pancreas, brain, but the highest expression occurred in normal ovary (FIG. 3D).
  • the blots were obtained from Clontech (CLONTECH Laboratories, Palo Alto, CA), and Northern blots performed as described above.
  • the inventors constructed the recombinant plasmids that express GST fusion proteins.
  • the NOEY2 cDNA fragment which included all four GTP binding domains has been obtained by PCRTM amplification and fused in-frame into pGEX-2T vectors to produce recombinant constructs GST-NOEY2 which expressed a fusion protein with M r 53,000 (53 kDa), of which 27 kDa was GST and 26 kDa was NOEY2 derived.
  • Large quantities of fusion protein were prepared and purified by preparative SDS-polyacrylamide gel electrophoresis (PAGE). Rabbit antiserum and monoclonal antibodies are being prepared by a standard protocol. Eight monoclonal antibodies against GST-NOEY2 have been obtained and used for detecting NOEY2 protein.
  • a 26 kDa protein were expressed by normal ovarian and breast epithelial cells, but could not be detected in ovarian and breast cancer cell lines (FIG. 3E).
  • Genomic DNA was isolated from confluent cell cultures by DNAzol reagent (GIBCO BRL Grand Island, NY). DNA samples (50 ⁇ g) were digested with restriction enzymes, separated by agarose gel electrophoresis and transferred to a hybond-nylon membrane. The Southern blot hybridization procedure was same as in Northern blot.
  • PI clones from the DuPont PI Genomic Library were screened with NOEY2 specific primer pairs Y2F3/B1-2 or Y2-19T/3Y2SP2 and used to determine their locations on human chromosome by fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • Probe DNA was extracted and labeled with digoxigenin-11-dUTP by nick translation.
  • the hybridized signal was detected by anti-digoxigenin conjugated with FITC.
  • the location of the probes was determined by digital image microscopy following FISH and localized by the fractional length from the p-terminus (Flpter). NOEY2 has been mapped to chromosome lp31 and Bacs obtained.
  • LOH at lp31 has been observed in a significant percent of breast and ovarian cancer (Kitayama et al, 1989). Using an intragenic TA dinucleotide repeat length polymo ⁇ hism as a marker, the inventors have detected LOH within the NOEY2 gene in 9 of 18 ovarian cancers (50%).
  • NOEY2 cDNA was excised from the EcoRI cloning sites from the Bluescript/ ⁇ ZapII® vector. This fragment was 1 Kb in size and included the ORF, it was inserted into the pcDNA3 neo eukaryotic expression vector (Invitrogen, San Diego, CA) in the sense and antisense orientation.
  • the constructs were transfected into three ovarian cancer cell lines (OVCA433, OVCA429, Hey), one breast cancer cell lines (SKBr3) and one lung cancer cell line (A549) by lipofectamin method (GIBCO BRL Grand Island, NY). As controls, similar amounts of carrier DNA and the vector DNA only were also transfected into cells. The mixtures of lipofectamin and constructs were exposed to the cells for six h at 37°C, then replaced by cell culture medium. After incubation for 48 h at 37°C, transfected cells were trypsinized and seeded into 100 mm dishes, select medium with G418 400 ⁇ g/ml to 1000 ⁇ g/ml was added to the cells. Replaced the select medium every four days. After 2-3 wk, transformed colonies began to appear, the dishes were stained by 0.1 %> coomassie blue in 30% methanol and 10% acetic acid.
  • NOEY2 sense constructs but not antisense constructs, strongly inhibited cyclin DI promoter activity when cotransfected with a plasmid containing the luciferase gene under the control of the cyclin DI promoter in Saos-2 and NIH3T3 as well as ovarian and breast cancer cell lines whose growth could be inhibited (FIG. 4B).
  • cyclin DI is required for Gl-S progression (Albanese et al, 1995)
  • the potent inhibition of the cyclin DI promoter activity by NOEY2 could contribute to the observed growth inhibition.
  • Cyclin DI expression is upregulated in 25-30% of ovarian cancers in the absence of gene amplification (Worsley et al, 1997).
  • EGF epidermal growth factor
  • insulin hydrocortisone
  • BPE bovine pituitary extract
  • the p 2l WAF1 C!P1 P rotein has been shown to arrest cell growth by inhibition of cyclin dependent kinases (Xiong et al, 1993). While p 2l WAF1 CIP1 i s p53 inducible gene (El-Deiry et al, 1993), induction of this inhibitor has also been observed in the absence of functional p53 (Michieli et al, 1994). Similarly, transfections of NOEY2 into the Hey (p53 wild type), SKBr3 (p53 mutant) and Saos-2 (p53 null) cells exerted p53 independent inhibition of cyclin DI promoter activity. These observations collectively suggest that NOEY2 functions as a negative regulator of cell growth probably through interaction with components of cell cycle control.
  • the genomic sequence of NOEY2 contains two exons and one intron.
  • the inventors have sequenced the hole coding region and 1.9 Kb upstream to identify putative mutations in NOEY2 using DNA from different cell lines.
  • Eight of the 18 tumor cell lines (SKOv3, OVCAR-3, OVCA429, OVCA432, BT20, MCF-7, MDA-MB-432 and SKBr3) have a heterozygous A to G substitution at -750 (44%), which is also presenting 5 of 36 primary ovarian cancer DNA samples (14%) and 20 of 100 DNA samples from normal peripheral leukocytes (20%).
  • sequence variation appears to be a functionally significant polymo ⁇ hism in that induced mutation of A to G at -750 reduces the activity of the NOEY2 promoter by more than 50% in OSE and NBE cells (FIG. 8).
  • sequence abnormalities have been found in the promote region of NDA-MB-468 with C(-13)G in one allele and G(+99)A in the other.
  • CAOv3 and BT20 each have a G(coding-231)A alteration encoding an Ala to Thr change, which also was found in 6 of 110 normal control DNA samples (5%).
  • NOEY2 Only one allele of NOEY2 was expressed in each of these cell lines as assessed by RT-PCRTM cDNA sequence analysis and by digestion with Hhal suggesting acquired methylation or germ line imprinting. Hypermethylation of CpG islands surrounding the TATA box was found in 2 or 8 breast cancer cell lines (MDA-MB4-35 and MDA-MB-453). Three OSE, 2 NBE DNA, 9 ovarian cancer and 4 breast cancer cell lines exhibited partial methylation at this site. Expression of only one NOEY2 allele may be important, given the high rate of LOH and the frequent occurrence of a functionally significant polymo ⁇ hism at -750 in the promoter region.
  • NOEY2 functions as a tumor suppressor.
  • EXAMPLE 2 ⁇ SIGNIFICANCE OF NOEY2 EXPRESSION LOSS IN OVARIAN CANCERS NOEY2 expression may be assessed at the level of message (with probes and primers already available) and protein (with antibodies developed as described below). Expression may be correlated with histology, stage, grade, outcome and response to chemotherapeutic agents.
  • DNA, RNA and protein may be obtained from primary ovarian epithelial cell cultures, ovarian cancer cell lines, primary ovarian cancer cells from patients' ascites and cryopreserved normal ovary and tumor tissues. Ascites tumor cells may be purified to >95% homogeneity using techniques developed in the inventors' laboratory (Hurteau et al, 1994).
  • NOEY2 cDNA probe was labeled with P-dCTP by random primer. Fifteen micrograms of total cellular RNA was separated in 1.2% formaldehyde-agarose gels and immobilized on a Hybond-N membrane by standard capillary transfer and UV crosslinking, and then prehybridized and hybridized to NOEY2 probe in 50% formamide. IX SSC. 10X Denhardt's solution, 10 mM EDTA, 0.1% SDS and 300 ⁇ g/ml denatured salmon sperm DNA at 42°C for 24 h. The blot was washed at 42°C in 0.1 x SSC 0.1% SDS before exposure. Hybridization of the same blot to a probe for 18S-rRNA indicates an equal amount of RNA in all lanes.
  • OSE medium MCDB 105/199 medium supplemented with 15% fetal calf serum and 10 ng/ml EGF.
  • Ovarian cancer cell lines were maintained as previously described (X et al, 1991).
  • NOEY2 cDNA (1 Kb) was released from the EcoRI cloning site in the
  • the HA-NO ⁇ Y2 and HA-Erk2 constructs were transfected into NIH3T3 cells using lipofectamine. After 24 h, cells were fixed with 4% paraformadehyde permeabilized with
  • Triton X-100 0.5% Triton X-100 and stained with an anti-p21 WAF1/CIP1 polyclonal antibody (c-19) (Santa Cruz) and anti-rabbit IgG FITC conjugate followed by subsequent incubation with an anti-HA- rhodamine conjugate (Boehringer Mannheim, Indianapolis, Indiana).
  • NOEY2 has the GenBank accession number U96750.
  • specific antibodies may be prepared against recombinantly derived NOEY2 fusion proteins.
  • the inventors have already constructed three recombinant plasmids that express GST fusion proteins. Three fragments (of 0.8 Kb, 0.6 Kb and 0.4 Kb) have been obtained from NOEY2 cDNA clones by PCRTM amplification. One included all four GTP binding domains, the second included the third and fourth domains and the third included the first and second domains.
  • the GST-NOEY2 fusion proteins were prepared and purified by preparative SDS-polyacrylamide gel electrophoresis (PAGE).
  • Anti-NOEY2 murine monoclonal antibodies may be prepared and screened using standard protocols which the inventors have previously used in development of OC125 (Bast et al, 1981) which binds CA125 antigen (Bast et al, 1983). Hybridomas may be screened against the immunizing GST-fusion protein and against GST by ELISA to identify those which specifically react with NOEY2. Polyclonal and monoclonal antibodies may be assessed for utility in immuno-precipitation, western blotting, immunohistochemistry and immunofluorescence utilizing the immunizing fusion protein as control.
  • STATISTICAL ANALYSIS Patient outcome may be characterized by one of the time-to-event variables 1) survival time or 2) disease-free survival (DFS) or by the binary variable indicating either 3) response or 4) partial response to chemotherapy (Cox, 1972; Modern Applied Statistics with S-Plus, 1994; Grambsch and Therneau, 1994; Harrington and Fleming, 1991).
  • Each of these evaluations may be carried out by regression analysis, with the patient outcome as the response variable in the regression model and NOEY2 included as a predictive covariate along with the established predictors disease stage, disease grade, amount of residual disease post surgery, and other molecular markers, including HER-2, ⁇ GY ,fins, and p53.
  • NOEY2 is recorded as an ordinal variable taking on the values 0,1,2,3 or 4, it may be evaluated first as a numerical covariate and subsequently as a categorical covariate in each regression analysis. For each patient outcome, specific questions to be addressed include whether NOEY2 per se is predictive, if so whether the effect of NOEY2 on patient outcomes survival of DFS changes over time, and whether any significant effect of NOEY2 persists in the presence of the established predictors noted above.
  • Relationships between pairs of covariates may be evaluated by computing standard Pearson correlations and Spearman rank correlations between numerical variables and constructing their smoothed scattergrams, by cross-tabulating categorical variables, and by carrying out Kruskal-Wallis or Wilcoxon-Mann- Whitney tests to assess the change of a numerical variable across a categorical variable.
  • Covariate effects on each of the time-to-event outcomes survival and DFS may be evaluated using the Cox proportional hazards regression model (Cox, 1972) and its extensions (Harrington and Fleming, 1991; Therneau, 1994). Goodness-of-fit may be evaluated using the methods of Therneau et al, (1990) and Lin (1991). Smoothed martingale residual plots may be used to determine if any of these markers have a possible "threshold effect" on survival or DFS (Kornblau et al, 1995; Hilsenbeck and Clark, 1996). Possible time-varying covariate effects may be identified and evaluated using the method of Grambsch and Therneau (Grambsch and Therneau, 1994).
  • PCRTM fragments with abnormal mobility were cloned into the PCR-Script Amp SK (+) cloning vector (Stratagene, LaJolla, CA). More than 10 clones of each sample were sequenced using an ABI PRISM 377 DNA automatic sequencer and a Big DyeTM terminator cycle sequencing kit. Upstream promoter region sequences (1.9 Kb) were amplified by four pairs of primers with a -21 Ml 3 tail. Purified PCRTM products were directly sequenced using -21 Ml 3 DyeTM primer cycle sequencing kit (PE Applied Biosystems). Methylation was measured using restriction enzymes Xbal/Sacll in CpG islands surrounding the TATA box by Southern blot analysis.
  • PROMOTER ACTIVITY ASSAY NOEY2 promoter fragment was amplified from genomic DNA by PCRTM and cloned into pGL2, a vector with luciferase reporter, its activity was tested by luciferase assay system (Promega, Madison, WI). A -750A to G mutant construct was made by site directed mutagenesis (Stratagene). Mutation was confirmed by sequencing.
  • the inventors expect that a loss of NOEY2 will be observed in a significant fraction of advanced stage and high grade ovarian carcinomas.
  • the inventors predict that NOEY2 may be lost in a smaller fraction of grade I and lower stage tumors.
  • the inventors predict that loss of NOEY2 may be a rare event in borderline tumors, but that this might correlate with high risk of recurrence.
  • the loss of NOEY2 may correlate with a poor prognosis in deeply invasive lesions.
  • NOEY2 may correlate with conventional risk factors
  • NOEY2 may be of even greater interest if its loss correlates with shortened survival but not with these conventional factors.
  • Loss of NOEY2 may or may not correlate with drug resistance. If this were the case for some drugs and not for others, the marker might prove particularly valuable in choosing primary therapeutic regimens for individual patients. Lack of a correlation with drug resistance would suggest that loss of NOEY2 was a marker for aggressive biological behavior. Interaction with other biological markers may be of particular interest. If NOEY2 inhibits signaling through the Ras pathway the inventors would anticipate amplification of the poor prognosis associated with persistence of EGFR, overexpression of HER-2 and novel expression offms.
  • NOEY2 has been mapped to lp31.
  • the inventors' data for 6 highly polymo ⁇ hic markers located at or near lp31 indicates that LOH occurred in 5 of 12 informative tumors from matched pairs of ovarian cancer and peripheral blood samples. This rate of 42% > loss is -I l l-
  • the inventors may undertake LOH analysis of the region around NOEY2 in DNA from ovarian cancers and normal peripheral blood leukocytes of the same patients.
  • the inventors may assess 100 matched pairs of tumor and normal peripheral blood leukocytes with histological and outcome evaluation.
  • the inventors may correlate LOH at lp31 with outcome as well as with stage, grade and histology. Southern blotting analysis has failed to detect abnormalities in the structure of the
  • NOEY2 gene in 6 of 6 ovarian cancer cell lines assessed.
  • the inventors may extend these studies to include the nine freshly isolated ovarian cancer ascites samples from patients which the inventors have already demonstrated do not express detectable amounts of NOEY2 RNA. If, as expected, the NOEY2 gene is intact in the freshly isolated samples as it is in the already characterized tumor cell lines, it is most likely that the loss of NOEY2 expression is due to changes in methylation status rather than due to deletion or to a transcriptional mechanism.
  • hypermethylation is a relatively frequent mechanism for loss of expression of tumor suppressor genes such as VHL, pi 6 and RB.
  • the inventors may thus assess the effect of decreasing methylation with 5-azacytidine or 5-aza-2-deoxycytidine on expression of NOEY2 in ovarian cancer cell lines.
  • the inventors may also assess whether the promoter for NOEY2 contains CpG islands and is hypermefhylated in ovarian cancers as compared to normal epithelium.
  • mutations might be found in the promoter region which may be sequenced in those cell lines and tumors where downregulation of NOEY2 expression cannot be attributed to other mechanisms.
  • LOH analysis is performed with 8 commercially available highly polymo ⁇ hic markers for lp31 using techniques published previously.
  • the analysis of the 100 samples utilizes a semi-automated microtiter system for analysis providing rapid throughput for the 100 samples and 8 primer pairs.
  • Southern blotting is performed using standard techniques on DNA isolated from highly purified (>95%) ovarian cancer cells isolated from the ascitic fluid of ovarian cancer patients and tumor cells isolated from solid tumors (samples with >80% tumor cells).
  • Hypermethylation of promoter regions may be sought in ovarian cancers that lack NOEY2 expression without LOH or an evident abnormality on Southern blots.
  • Ovarian cancer cell lines demonstrated not to express NOEY2 and shown to have a relatively intact NOEY2 gene on Southern analysis are cultured for 48 h to 2 wk in 5-azacytidine (5 M) or 5-aza-2- deoxycytidine (.75 M) to determine if this alters the expression of NOEY2 as assessed by Northern blot analysis.
  • Analysis of the NOEY2 promoter is conducted to determine whether the promoter for NOEY2 contains CpG islands.
  • the methylation status of the promoter for NOEY2 in ovarian cancer cell lines is assessed by restriction analysis with methylation-sensitive restriction enzymes including Hhal, Not! and S cII. Alterations in enzyme sensitivity may be due to changes in methylation, and this is analyzed by comparing the restriction patterns with those of normal ovarian epithelial cells which express NOEY2 and with ovarian cancer cell lines incubated with 5-azacytidine or 5-aza-2-deoxycytidine.
  • ovarian cancer cells may be isolated and purified from ovarian cancer patient's ascites. Ovarian cancer cells can be purified to over 95% homogeneity (Hurteau et al, 1994) and may routinely survive in culture for at least one month. Such cells are assessed for the effect of 5-azacytidine or 5-aza-2-deoxycytidine on NOEY2 expression and restriction analysis with methylation sensitive restriction enzymes including Hhal, Notl and Sac ⁇ l as described for cell lines above.
  • a method for methylation detection may be utilized which depends upon the chemical modification of cytosine to uracil by bisulfite treatment (Frommer et al, 1992). This technique is less sensitive to contamination of tumor by normal cells and is more sensitive in detecting hypermethylation than is the restriction enzyme approach described above.
  • genomic DNA is bisulfite treated and fragments from within the promoter containing region cloned using PCRTM and the TA vector and then subjected to sequence analysis.
  • sequence analysis may reveal the conversion of unmethylated cytosines to thymidines, while methylated cytosines may appear as cytosines in the sequencing analysis. Sequencing of a number of TA vector plasmids from tumors may demonstrate the frequency of methylation of the NOEY2 promoter.
  • Transcriptional regulation may be evaluated in those cell lines and tumor specimens where loss of NOEY2 expression cannot be attributed to other mechanisms. At least 500 bp of the NOEY2 promoter may be cloned from the BACs already identified that contain the NOEY2 coding sequence. The promoter may be ligated to luciferase as a reporter gene (Gum et al, 1996). Transient expression is sought after transfection of ovarian cancer cell lines that lack NOEY2 expression and, as a positive control, normal ovarian surface epithelial cells that express NOEY2.
  • Transcriptional regulation may be the least frequently occurring abnormality, however, cell lines that exhibit such an abnormality are valuable for elucidating mechanisms of physiologic as well as pathologic regulation of NOEY2 expression.
  • NOEY2 expression is consistently lost from ovarian cancer cell lines and from freshly isolated cancer cells from patient's ascites. Further, data indicate that expression of NOEY2 decreases clonogenic activity of ovarian and breast cancer cells in a transient transfection assay. This suggests that NOEY2 may alter characteristics of ovarian cancer cells which could lead to initiation or progression of ovarian cancer.
  • Ras and Rap proteins require appropriate localization and an intact effector domain to mediate their functions. Mutagenesis of any of the amino acids in the CAAX box or the YDPTIEDSY domain impairs the ability of Ras to transform cells.
  • Conditional expression of normal NOEY2 is used to test whether upregulation of NOEY2 inhibits growth, blocks invasion, reduces metastatic potential, or decreases angiogenesis, and uses a dominant negative NOEY2 (N17-NOEY2 modeled on other dominant negative small G protein constructs) to determine whether downregulation of NOEY2 activity may stimulate growth, increase invasive potential, augment metastatic potential, or enhance angiogenesis.
  • a dominant negative NOEY2 N17-NOEY2 modeled on other dominant negative small G protein constructs
  • the inventors assess whether expression of NOEY2 may alter cell cycle progression or direct cells to apoptosis.
  • ovarian cancer growth inhibition by a number of mediators including heregulin can require an intact p53 pathway or an intact RB pathway. Approximately 50%) of all ovarian cancers have mutations or deletions in p53. All of the ovarian cancer cell lines thus far assessed for growth inhibition by NOEY2 contained normal p53. Although mutations in RB or pi 6 are rare in freshly isolated ovarian cancer cells, the inventors have identified ovarian cancer cell lines with deletions or mutations in p53, deletions in pi 6 and cell lines with deletions in RB.
  • the effector domain of Ras is required for the transforming activity of the protooncogene.
  • the effector domain of Ras and Rap family members is critical for Ras and Rap functions.
  • the effector domain of NOEY2 differs from that of Ras and Rap family members. Site directed mutagenesis may be utilized to convert the effector domain of NOEY2 to the identical sequence to that found in Ras and Rap family members.
  • NOEY2 normal and epitope-tagged NOEY2 has been inserted into CMV and SV40 promoter driven expression constructs.
  • NOEY2 gene has been epitope tagged with an hemagglutinin (HA) epitope using a triple HA repeat plasmid.
  • the HA epitope was added to the N terminus of NOEY2 so as to be distant from the potential myristylation site at the C terminus of NOEY2.
  • the Tet-Off and Tet-On Gene Expression System provides regulated, high-level gene expression as initially described by Gossen and Bujard (1992). In the first transfection, the Tet-On regulatory protein is introduced into ovarian cancer cell lines by transfection of a "regulator plasmid"-pTet-On (cells already isolated).
  • NOEY2 and epitope tagged NOEY2 have already been inserted into the pTRE (plasmid created) which may be introduced into cells expressing the Tet On plasmid to create double-stable Tet-On cell lines which may express NOEY2 only in the presence of tetracycline.
  • NOEY2 gene expression should be negligible in the absence of tetracycline and induced by the addition of tetracycline.
  • the Tet-on system has the advantage over most inducible systems in having very low levels of non-induced expression (leakiness) and in being compatible with induction both in vitro and in vivo.
  • Mutant or normal NOEY2 may be transfected into ovarian cancer cells along with a hygromycin resistance plasmid. Cells may be incubated with hygromycin for 2-3 wk and colonies assessed by staining with 1% Coomassie blue in 30%> methanol and 10%) acetic acid.
  • Proliferative capacity may be judged by ⁇ H-thymidine inco ⁇ oration, growth by MTT assay and clonogenicity by growth in soft agar in the presence and absence of tetracycline. Invasion of matrigel membranes may be studied using techniques already established in the inventors' laboratory (Berchuck et al, 1992). Apoptosis may be evaluated by mo ⁇ hology, free DNA ends (Apoptag kit), DNA ladders and loss of membrane asymmetry (Annexin V staining).
  • the parental ovarian cancer cell lines-SKOv3 and Hey are tumorigenic in nude mice (HEY forms subcutaneous tumors whereas the HEY-A8 cell line forms intraperitoneally tumors and metastasizes).
  • Five to 10 x 10" stably transfected tumor cells from each cell line may be injected subcutaneously and intraperitoneally into athymic nude mice. All recipients should develop tumors within 2 wk. If the behavior of transfected cells reflects the parental phenotype, tumor cells may form nodules, ascites, and metastasize to the lungs where nodules can be detected grossly and by histologic examination.
  • Doxycychne when doxycychne is given to the mice at a dosage sufficient to establish an appropriate doxycychne concentration, NOEY2 gene expression in the tumor cells should be upregulated and growth inhibited.
  • Doxycychne can be delivered either orally as a glucose suspension or optimally as a long release pellet (Innovative Research). Subcutaneous tumors may be measured twice a wk and animals examined every other day for ascites.
  • NOEY2 inhibits colony forming activity of breast and ovarian cancer cell lines.
  • the effects of NOEY2 may be expressed at many levels by inhibiting proliferation, colony formation, anchorage independent growth, invasiveness and ability to form tumors in nude mice.
  • the global inhibitory activity of NOEY2 may extend to an ability to block production of growth factors and angiogenic factors such as VEGF. NOEY2 may thus decrease the ability of tumors to grow in the peritoneal cavity, to metastasize and to enlarge due to lack of neovascularization.
  • NOEY2 appears to function as a tumor suppressor in ovarian cancer.
  • NOEY2 could function as a tumor suppressor by interfering with signal transduction from surface receptors, by interfering with signaling cascades, or by altering the function or expression of proteins required for cell cycle progression.
  • NOEY2 may target cells for apoptosis. This specific aim may attempt to identify the site at which NOEY2 mediates it tumor suppressor activity.
  • the product of the NOEY2 gene may act as an antagonist of Ras p21 protein signaling.
  • Ras provides a convergence point for signal transduction induced by tyrosine kinase linked and G protein linked receptors.
  • a major pathway activated by p21-Ras is the Ras-Raf-MEK-ERK (MAPK) module which plays an essential role in cell growth and differentiation. Due to its structural similarities to p21-Ras, NOEY2 protein may compete for the upstream or downstream regulators and effectors of Ras. This is a suggested mechanism of action for the ability of Rap family members to decrease Ras-mediated signaling.
  • the product of the NOEY2 gene may act as an agonist of the JNK pathway.
  • the JNK pathway includes a similar G protein-cascade module, RAC-MEKK1-SEK/MKK4-JNK/SAPK.
  • MAP kinases JNK has been linked to stress response, growth inhibition and programmed cell death. Since the JNK pathway is a negative regulator of cell proliferation, an agonistic activity of NOEY2 in this pathway could explain the tumor suppressor-like activity of NOEY2. Thus it is possible that NOEY2 protein inhibits cell growth through activation of the JNK pathway.
  • NOEY2 decreases colony forming activity, it is likely that it inhibits cell cycle progression.
  • the inventors may assess whether intact p53, pi 6 or RB are required for NOEY2 to inhibit colony forming cell activity. If the p53 or pl6/RB pathways are obligatory for NOEY2 inhibition of colony forming cell activity, the inventors may assess the effect of NOEY2 on activation of these pathways. As an alternative to inhibition of cell cycle progression, NOEY2 may decrease colony forming cell activity through induction of apoptosis.
  • NOEY2 increases the rate of apoptosis
  • the inventors may assess the ability of NOEY2 to alter expression of members of the Bcl-2 family of proteins which play a major role in the regulation of programmed cell death in ovarian cancer cells according to the inventors' data.
  • Small G proteins are active in the GTP bound state and inactive in the GDP bound state.
  • the localization of Ras and other small G proteins through myristylation is critical for their function.
  • NOEY2 may function at a number of levels in suppressing tumorigenesis, it may be interesting to determine whether NOEY2 inhibits the transforming activity of growth factor receptors (EGFR, HER-2), intracellular tyrosine kinases (src), activated Ras and downstream mediators of Ras (RAF).
  • EGFR growth factor receptors
  • HER-2 growth factor receptors
  • src intracellular tyrosine kinases
  • RAF downstream mediators of Ras
  • Expression plasmids carrying VI 2 Ras, v-raf or MEK may be cotransfected with NOEY2 or an empty vector.
  • MAPK activity in transfected cells may then be determined using an epitope-tagged cotransfected MAPK.
  • a complete or partial inhibition of Ras-, Raf- or MEK-stimulated MAPK activation by NOEY2 would be expected if NOEY2 inhibits the Ras signaling cascade.
  • the RAC and CDC42 small G proteins activate the JNK signaling pathway which negatively regulates cell proliferation targeting cells to programmed cell death.
  • the effector domain of NOEY2 has a sequence which is similar to that of RAC and CDC42, the inventors may assess whether NOEY2 activates the JNK kinases.
  • the inventors may also assess whether NOEY2 is required for activation of the JNK kinases by TNF, IL1, FAS, lysophospholipids, and protein synthesis inhibitors (anisomycin) all of which the inventors have shown activate JNK kinases in ovarian cancer cells or activate ovarian cancer cells and have been demonstrated to activate JNK kinases.
  • the inventors may assess whether intact pi 6 or intact RB is required for NOEY2 to mediate inhibition of colony formation. If pi 6 or intact RB is required for the inhibition of cell proliferation, the inventors may assess the effect of NOEY2 on pi 6 and cyclin DI levels, on CDK4 kinase activity and on RB phosphorylation status. The inventors may also assess whether normal p53 is required for NOEY2 to mediate inhibition of colony formation. If p53 is required for NOEY2 to mediate inhibition of colony formation, the inventors may assess whether NOEY2 alters expression or stability of p3, expression of cyclin DI, CDK2 kinase activity and expression of the P21/WAF1/CIP mediator of p3 action.
  • the inventors have developed polyclonal (9613, 9617) and eight monoclonal (17G6, 15E11, 12C7, 6B4, 6D2, 12A8, 3H2 and 16C6) antibodies to NOEY2 with techniques that the inventors have successfully utilized in the past.
  • the inventors have tagged NOEY2 with HA- epitope allowing the inventors to distinguish transfected NOEY2 from endogenous NOEY2.
  • polyclonal or monoclonal antibodies specific to NOEY2 are available, the inventors may utilize transfection of epitope-tagged NOEY2 to demonstrate function.
  • the HA antibody (for which the inventors have access to the hybridoma) immunoprecipitates tagged proteins and works well in Western blotting.
  • GTP/GDP ratios are assessed using 32 P0 -loaded cells as reported previously for determining Ras GTP/GDP ratios (Kruk et al, 1990). NOEY2 may be immunoprecipitated and washed. GTP and GDP may be separated by thin layer chromatography and ⁇ - ⁇ P-containing GTP and GDP identified by autoradiography and comparison with ninhydrin stained standards.
  • Normal ovarian epithelial cells which contain more NOEY2 protein than ovarian cancer cell lines may be used.
  • Cells may be labeled with 3 s-methionine for 3 h and nuclear, cytoplasmic and membrane fractions prepared by differential centrifugation. Equivalent amounts of each sample may be immunoprecipitated and analyzed by SDS-PAGE.
  • NOEY2 may be western blotted to determine localization. If insufficient NOEY2 is present to allow direct western blotting, NOEY2 may be immunoprecipitated from each cellular fraction and localization determined by western blotting of each fraction.
  • NOEY2 may be identified by immunoprecipitation of transfected HA epitope tagged NOEY2 until such time as NOEY2 antibodies are available. Normal ovarian epithelial cells can be effectively transfected.
  • HA may be immunolocalized using confocal microscopy and immunoelectron microscopy.
  • the HA antibody and epitope tagged NOEY2 cannot be utilized for this pu ⁇ ose due to cross-reactivity of the HA antibody with cellular proteins.
  • antisense NOEY2 and dominant negative NOEY2 may be cotransfected at a
  • NIH 3T3 cells 5:1 ratio with activated erbB, HER-2, src, Ras and RAF into NIH 3T3 cells to ensure that NOEY2 is present in all transfected cells.
  • the effect of NOEY2 on focus forming activity induced by each oncogene may then be assessed.
  • the inventors have NIH3T3 cells in the laboratory with a low baseline focus forming rate which are appropriate for this assay.
  • NIH 3T3 cells constitutively overexpressing myc are available, which may be used to determine whether NOEY2 may inhibit the focus forming activity of these oncogenes in the presence of high levels of myc.
  • NOEY2 The ability of NOEY2 to alter activation of Ras, MAPK, or JNK may be assessed by cotransfection of NOEY2, antisense NOEY2, and dominant negative NOEY2 (not epitope tagged) and an epitope tagged target.
  • NOEY2 antisense NOEY2
  • dominant negative NOEY2 not epitope tagged
  • an epitope tagged target Alternatively if conditional tetracycline-induced NOEY2 containing cell lines are developed, the inventors may study endogenous targets with and without Tet induction of NOEY2 expression.
  • the inventors may also assess whether NOEY2 affects the ability of activation of tyrosine kinase linked receptors (EGF and heregulin), activation of protein kinase C (phorbol esters) and G protein linked receptors (lysophosphatidic acid) to stimulate the activity of these signaling molecules.
  • GEF and heregulin tyrosine kinase linked receptors
  • protein kinase C phorbol esters
  • G protein linked receptors lysophosphatidic acid
  • GTP/GDP ratios on Ras may be determined as described above with 3 p labeling and immunoprecipitation of epitope-tagged Ras with the anti-HA antibody or in conditional NOEY2 expressing cells with the Yl 3-259 anti-Ras antibody (hybridoma in house).
  • NOEY2 The effect of NOEY2 on MAPK activation may be assessed by four assays: 1) gel mobility shift. 2) Western blotting with an activation specific epitope antibody (UBI) 3) in gel kinase assay (the epitope tagged MAPK runs at a different size from the native MAPK) and 4) in vitro kinase using MBP as a substrate (Xu et al, 1994).
  • UBI activation specific epitope antibody
  • JNK ASSAY JNK activity may be determined by in vitro kinase assay using a GST-N-terminal portion fusion protein of Jun as substrate. The inventors may also assess whether expression of NOEY2 or of dominant negative (N17 NOEY2) may alter anisomycin, TNF, IL1 or FAS induced activation of JNK.
  • conditional tetracycline-induced NOEY2 containing cell lines are developed, the inventors may study endogenous cyclin DI levels with and without Tet induction of NOEY2 expression. Alternatively, if conditional NOEY2 containing cell lines are not available, the inventors may utilize cotransfection of NOEY2 and a cyclin DI luciferase expression construct as a reporter.
  • CDK4 and CDK2 kinase activity may be assessed following immunoprecipitation using recombinant RB (UBI) as a substrate.
  • p21/WAFl/CIPl and pl6 levels may be assessed using western blotting with UBI antibodies.
  • RB phosphorylation may be measured by gel shift assay using RB specific antibodies (UBI).
  • p53 levels may be assessed by immunohistochemistry and western blotting. Although p53 levels are normally low, they can be increased by transcriptional or post transcriptional mechanisms which can be detected by an increase in levels as detected by these methods. The inventors may assess the effect of NOEY2 on expression of these proteins by western blotting or by immunoprecipitation followed by western blotting where protein levels are low.
  • NOEY2 may have constitutively high GTP/GDP ratios.
  • NOEY2 contains a CAAX Box
  • the inventors predict that NOEY2 may be primarily a membrane associated protein.
  • the inventors have demonstrated that NOEY2 inhibits the growth of ovarian cancer cells which contain activated or mutant Ras.
  • the inventors predict that NOEY2 may function similar to Rap family members and may inhibit activation of the Ras by tyrosine kinase, and G protein linked receptors.
  • NOEY2 may inhibit MAPK activation induced by tyrosine kinase and G protein linked receptors and by activated Ras.
  • NOEY2 may not inhibit activation of the Ras pathway by activated RAF or MEK.
  • the inventors predict that NOEY2 may function at the GI phase of the cell cycle.
  • the inventors expect that NOEY2 may block cell cycle progression in GI by inhibiting the pathway leading to RB phosphorylation. Without further data, it is impossible to predict at which level in the CDI/CDK/Cyclin RB cascade that be regulated by NOEY2.
  • FIG. 7 shows the genomic structure of the NOEY2 locus. The genomic sequence of this region appears below:
  • RNA moiety of ribonuclease P is the catalytic subunit of the enzyme
  • Kruk, Maines-Bandiera, Auersperg "A simplified method to culture human ovarian surface epithelium," Lab. Invest, 63(1):132-136, 1990. Kuby, "Immunology” 2nd Edition. W.H. Freeman & Company, New York, 1994. Kunkel, Roberts, Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection,” Methods Enzymol, 154:367-382, 1987. Kwoh et ⁇ /., Proc. Natl. Acad. Sci, USA, 86(4): 1173-1 177, 1989. Kyte and Doolittle, J. Mol Biol, 157:105-132, 1982.
  • Perricaudet "Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo," Gene, 101 :195-202, 1991.
  • Temin "Retrovirus vectors for gene transfer: Efficient integration into and expression of exogenous DNA in vertebrate cell genome," In: Gene transfer, Kucherlapati R, ed., New York: Plenum Press, pp. 149-188, 1986.
  • HIV-1 infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expression an HIV-1 RNA-specific ribozyme
  • J. Virol, 65(10):5531- 5534, 1991 conferred by using retroviral vectors expression an HIV-1 RNA-specific ribozyme
  • Yu Ojwang, Yamada, Hampel, Rapapport, Looney, Wong-Staal, "A hai ⁇ in ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1," Proc. Natl. Acad. Sci. USA, 90:6340-6344, 1993. Yu, Henry, Xu, Hamilton, “Expression of a murine cytomegalovirus early and late protein in latently infected mice," J. Infectious Diseases, 172:371-379, 1995a. Yu, Matin, Xia, Sorgi, Huang, Hung, "Liposome-mediated in vivo El A gene transfer suppressed dissemination of ovarian cancer cells that overexpress HER-2/neu,"
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Thr Thr Glu Lys Leu Leu Asp Lys Cys lie lie Met 220 225 GCCTTAAGAG CCAGCTCTTC CTATCCTGTA GCGTGTAGAA AACGTGGACT CATTTCACTA 903
  • Arg Lys lie Arg Asp Tyr Arg Val Val Val Val Gly Thr Ala Gly Val 35 40 . 45
  • CAGCCCCTCA CAGCTGGTTT NTTACCANGT ATTGCGCAAG CGGAATTTAT GCNTGTTACC 2040
  • TATATATATA TATATATATA TATATATATA TATATCTTTT TTTTTTTTTT TTTTTTTT 4740 TTTTTTACTC CACTGTCATT GTGACTAAGG ATTCATGAAC TAAGACCCCT CCCTCAGCTT 4800
  • CTGTTCAGTC CACTTCAATT AAAATCTTAA TTTTACAAGC GAGGAAATGA GAGTGTTTCT 5100

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Abstract

L'invention porte sur des compositions et méthodes utilisant un nouveau gène suppresseur de tumeurs, le NOEY2 qui est exprimé par les cellules épithéliales superficielles saines des ovaires et du sein, mais régulièrement absent ou sous-produit dans les cellules cancéreuses des ovaires et du sein. L'invention porte également sur des compositions de polynucléotides comprenant un gène NOEY2 provenant de mammifères et sur des polypeptides codés par ces séquences d'acide nucléique. L'invention porte en outre sur des procédés de préparation des polypeptides NOEY2, des cellules hôtes transformées, et des anticorps réagissant avec les polypeptides NOEY2. Dans certaines de ses réalisions, l'invention porte sur des méthodes de diagnostic et de traitement du cancer, et des méthodes d'identification des compositions de polynucléotides et de polypeptides apparentés au NOEY2.
PCT/US1998/005723 1997-03-21 1998-03-20 Nouvelles compositions de genes noey2 et leurs utilisations WO1998042830A2 (fr)

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EP98911977A EP0988376A2 (fr) 1997-03-21 1998-03-20 Nouvelles compositions de genes noey2 et leurs utilisations
US10/166,325 US7183401B1 (en) 1997-03-21 1998-03-20 NOEY2 gene compositions and methods of use
JP54590098A JP2001517954A (ja) 1997-03-21 1998-03-20 Ｎｏｅｙ２遺伝子組成物およびその使用方法
CA002284685A CA2284685A1 (fr) 1997-03-21 1998-03-20 Nouvelles compositions de genes noey2 et leurs utilisations
AU65805/98A AU6580598A (en) 1997-03-21 1998-03-20 (noey2) gene compositions and methods of use

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WO2000037141A1 (fr) * 1998-12-22 2000-06-29 Warner-Lambert Company Chimiotherapie combinee
WO2003057156A2 (fr) * 2001-12-31 2003-07-17 Strang Cancer Prevention Center Lignees cellulaires epitheliales issues de souris knock-out et leurs methodes d'utilisation
JP2004500852A (ja) * 2000-05-19 2004-01-15 エフ．　ホフマン−ラ　ロシュ　アーゲー ヒト腫瘍細胞においてダウンレギュレートされる核酸の使用による、試料の殺腫瘍能を決定するためのプロセス
CN104388540A (zh) * 2014-10-15 2015-03-04 上海赛安生物医药科技有限公司 Noey2基因突变检测体系及其试剂盒

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037141A1 (fr) * 1998-12-22 2000-06-29 Warner-Lambert Company Chimiotherapie combinee
JP2004500852A (ja) * 2000-05-19 2004-01-15 エフ．　ホフマン−ラ　ロシュ　アーゲー ヒト腫瘍細胞においてダウンレギュレートされる核酸の使用による、試料の殺腫瘍能を決定するためのプロセス
WO2003057156A2 (fr) * 2001-12-31 2003-07-17 Strang Cancer Prevention Center Lignees cellulaires epitheliales issues de souris knock-out et leurs methodes d'utilisation
WO2003057156A3 (fr) * 2001-12-31 2004-05-06 Strang Cancer Prevention Ct Lignees cellulaires epitheliales issues de souris knock-out et leurs methodes d'utilisation
CN104388540A (zh) * 2014-10-15 2015-03-04 上海赛安生物医药科技有限公司 Noey2基因突变检测体系及其试剂盒
CN104388540B (zh) * 2014-10-15 2016-08-24 上海赛安生物医药科技有限公司 Noey2基因突变检测体系及其试剂盒

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WO1998042830A3 (fr) 1998-12-23
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